Monthly Archives: July 2022

A genetic setup to investigate boundary function in vivo

We previously demonstrated that a 150-kilobase (kb) region, the EP boundary, is sufficient to segregate the regulatory activities of the Epha4 and Pax3 TADs10 (Extended Data Figs. 1 and 2). The DelB background carries a large deletion that removes this boundary region, and the Epha4 gene, resulting in the ectopic interaction between the Epha4 limb enhancers and the Pax3 gene. This causes Pax3 misexpression and the shortening of fingers (brachydactyly) in mice and in human patients. In contrast, the DelBs background carries a similar deletion but not affecting the EP boundary, which maintains the Epha4 and Pax3 TADs and confines the Epha4 enhancers within their own regulatory domain (Fig. 1a and Extended Data Fig. 1).

a, cHi-C maps from E11.5 distal limbs from DelBs mutants at 10-kb resolution. Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). The red rectangle marks the EP boundary region. Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Cen, Centromeric; Tel, Telomeric. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Below, Lac-Z staining (left) and WISH (right) of E11.5 mouse forelimbs show activation pattern of Epha4 enhancers and Pax3 expression, respectively. b, CTCF ChIPseq track from E11.5 mouse distal limbs. Schematic shows CBS orientation. c, Insulation score values. The gray dot represents the local minima of the insulation score at the EP boundary. BS, boundary score. d, Relationship between BS and the number of CBSs (data from ref. 26). The boxes in the boxplots indicate the median and the first and third quartiles (Q1 and Q3). Whiskers extend to the last observation within 1.5 times the interquartile range below and above Q1 and Q3, respectively. The rest of the observations, including maxima and minima, are shown as outliers. N=8,127 insulation minima found in mESC Hi-C matrices. e, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Note Pax3 misexpression on the distal anterior region in R1, F1 and F2 mutants (white arrowheads). Scale bar, 250m. f, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (two-sided t-test *P0.05; NS, nonsignificant; P values from left to right: DelBs versus R1: 0.02; DelBs versus R2: 0.11; DelBs versus F1: 0.02; DelBs versus R3: 0.23; DelBs versus F2: 0.02; DelBs versus R4: 0.73). Cen, Centromeric; Tel, Telomeric.

To characterize the EP boundary in vivo, we performed CTCF ChIPseq on developing limbs. This analysis revealed the presence of six clustered CBSs at the EP boundary region (Fig. 1a,b and Extended Data Fig. 2), a profile that is conserved across tissues25,26. CTCF motif analyses confirmed the divergent orientation of these sites, a signature of TAD boundaries, with four CBSs in reverse (R) and two in forward orientation (F). Other features associated with boundaries, such as active transcription or housekeeping genes, were not found in the region27 (Extended Data Fig. 3). cHi-C data from DelBs stage E11.5 distal limbs28 revealed chromatin loops connecting the two forward-oriented CBSs (F1 and F2) with the telomeric boundary of the Pax3 TAD, and the centromeric boundary of the Epha4 TAD with the reverse-oriented CBSs R1, R2 and R3 (Fig. 1a,b). However, the close genomic distances between R2 and F1 and between R3 and F2 preclude the unambiguous assignment of loops to specific sites. RAD21 (cohesin subunit) ChIPseq experiments in E11.5 distal limbs revealed that R1, F1 and F2, as well as R2 and R3 to a lesser degree, are bound by cohesin (Extended Data Fig. 3), an essential component for the formation of chromatin loops21,29,30. These results delineate the EP element as a prototypical boundary region with insulating properties likely encoded and controlled by CBSs.

Boundary regions are predominantly composed of CBS clusters31, suggesting that the number of sites might be relevant for their function. We explored this by calculating boundary scores32 on available Hi-C maps26, and categorizing boundaries according to CBS number. We observe that boundary scores increase monotonically with CBS number, reaching a stabilization at ten CBSs (Fig. 1d). According to this distribution, the EP boundary falls within a range where its function might be sensitive to alterations on CBS number. To test this, we employed a mouse homozygous embryonic stem cell (mESC) line for the DelBs background28, which we edited to generate individual homozygous deletions for each of the six CBSs of the EP boundary region (Supplementary Fig. 1). ChIPseq experiments revealed that the disruption of the binding motif was sufficient to abolish CTCF recruitment (Supplementary Fig. 2). Subsequently, we employed tetraploid complementation assays to generate mutant embryos and measure the functional consequences of these deletions in vivo33,34.

Whole-mount in situ hybridization (WISH) on E11.5 mutant embryos revealed that the insulation function of the EP boundary can be sensitive to individual CBS perturbations (Fig. 1e). However, this effect was restricted to CBSs displaying prominent RAD21 binding (R1, F1 and F2) (Extended Data Fig. 3). The altered boundary function was evidenced by Pax3 misexpression on a reduced area of the anterior limb, while the expression domains in other tissues remained unaltered (Supplementary Fig. 3). The disruption of the other CBSs (R2, R3 and R4) did not alter Pax3 expression, demonstrating that the EP boundary can also preserve its function despite a reduction in CBS number.

To quantify Pax3 misexpression, we performed quantitative PCR (qPCR) in E11.5 forelimbs. Similarly, we observed a modest, but significant, upregulation in R1, F1 and F2 mutants (Fig. 1f). Importantly, the functionality of individual CBSs is not strictly correlated with CTCF occupancy as the deletion of R3, displaying the highest levels of CTCF binding among the cluster (Fig. 1b and Extended Data Fig. 3), does not result in measurable transcriptional changes (Fig. 1f). Thus, while CBS number influences insulation, the characteristics of individual sites are major determinants of boundary function.

To explore CBS cooperation, we retargeted our R1 mESC line to generate double knockout mutants with different (R1+F2) or identical CBS orientations (F1 and F2 in F-all) (Fig. 2a). WISH revealed an expanded Pax3 misexpression towards the posterior region of the limb, demonstrating that the EP boundary is compromised in both mutants. Next, we determined the nature of CBS cooperation by qPCR. These experiments revealed that, in both mutants, Pax3 misexpression exceeded the summed expression levels from the corresponding individual deletions (Fig. 2b). These negative epistatic effects indicate that CBSs are partially redundant, compensating for the absence of each other.

a, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Note increased Pax3 misexpression towards the posterior regions of the limb. Scale bar, 250m. b, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (**t-test **P0.01; R1+F2 versus F-all: 0.008). c, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. d, Insulation score values. Lines represent indicated mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. e, Virtual 4C profiles for the genomic region displayed in c (viewpoint in Pax3). The light-gray rectangle highlights the Epha4 enhancer region. Note increased interactions between the Pax3 promoter and the Epha4 enhancer in R1+F2 and F-all (purple and orange) compared with DelBs mutants (gray).

To gain insights on the mechanisms of CBS cooperation, we generated cHi-C maps of the EP locus from E11.5 distal limbs (Fig. 2c and Supplementary Fig. 4). Maps from R1+F2 embryos denoted a clear partition between the EphaA4 and Pax3 TADs, analogous to DelBs control mutants (Fig. 2c). However, subtraction maps revealed decreased intra-TAD interactions for the Epha4 and Pax3 TADs, and a concomitant increase in inter-TAD interactions. In addition, we observed the appearance of a loop connecting the outer boundaries of the Epha4 and Pax3 TADs (meta-TAD loop; Extended Data Fig. 4)35. Accordingly, the boundary score of the EP boundary in R1+F2 mutants was decreased, reflecting a weakened structural insulation (Fig. 2d). Virtual Circular Chromosome Conformation Capture (4C) profiles revealed increased chromatin interactions between the Pax3 promoter and the Epha4 limb enhancers (Fig. 2e), consistent with the upregulation of Pax3. In addition, two of the chromatin loops that connect the EP boundary and the telomeric boundary were abolished, due to the deletion of the F2 anchor and the associated loss of RAD21 (Fig. 2c and Extended Data Figs. 4 and 5). Consequently, the adjacent chromatin loop exhibited a compensatory effect, with increased interactions mediated by the F1 anchor, consistent with higher RAD21 occupancy (Extended Data Figs. 4 and 5). At the centromeric site, the deletion of R1 causes the relocation of the loop anchor towards an adjacent region containing a reverse-oriented (R2) and the only remaining forward CBS (F1). While the loop extrusion model would predict a stabilization at a reverse CBS15,16, the short genomic distance between R2 and F1 precludes an unambiguous assignment of the loop anchor. We also observed increased contacts at R3 and R4, suggesting that these sites are functionally redundant.

Then, we examined cHi-C maps from F-all mutants, which display a more pronounced Pax3 misexpression (Fig. 2b). Interaction maps revealed a partial fusion of the Epha4 and Pax3 domains (Fig. 2c), accompanied by a notable decrease of the boundary score (Fig. 2d). Virtual 4C profiles confirmed increased interactions between Pax3 and the Epha4 enhancers in F-all compared with R1+F2 mutants, in agreement with the more pronounced Pax3 upregulation (Fig. 2e). The deletion of all CBSs with forward orientation abolishes the chromatin loops connecting with the telomeric Pax3 boundary (Fig. 2c and Extended Data Fig. 4). Towards the centromeric side, R1 maintains RAD21 binding and its chromatin loop with the centromeric Epha4 boundary (Extended Data Figs. 4 and 5). However, other chromatin loops are still discernible and anchored by the R3 and R4 sites, confirming that these sites perform distinct yet partially overlapping functions. These results demonstrate that CBSs can cooperate but also partially compensate for the absence of each other, conferring functional robustness to boundaries.

Chromatin loops are predominantly anchored by CBS pairs with convergent motif orientation14,36. Intriguingly, we observed that the combined F1 and F2 deletion (F-all) not only disrupts the loops in the expected orientation (telomeric), but also impacts the centromeric one, as observed in the subtraction maps (Fig. 2c). This effect is noticeable at the R2/F1 site, which was associated with a centromeric chromatin loop in the DelBs background (Fig. 1a). This demonstrates that the main loop anchor point was not the R2 but the F1 site (Extended Data Fig. 4), suggesting that this CBS can form loops in a nonconvergent orientation. Such mechanism is described by the loop extrusion model, which predicts that loops could create steric impediments that might prevent additional cohesin complexes from sliding through anchor sites15,16. This effect would stabilize these additional cohesin complexes, resulting in the establishment of simultaneous and paired nonconvergent and convergent loops (Fig. 3a).

a, Schematic of a convergent loop that indirectly generates a nonconvergent loop in the opposite direction. b, Percentage of loop anchors establishing bidirectional loops (n=12,635 loops from mESCs from ref. 26). Anchor categories: convergent-only (only CBSs oriented in the same direction as their anchored loops, n=7,769), nonconvergent (anchor loops in a direction for which they lack a directional CBS, n=960) and no-CTCF (no CBS, n=3,906). c, Loop strengths in pairs of convergent/nonconvergent loops classified into Non-conv.-associated (nonconvergent loop sharing the nonconvergent anchor with a convergent loop in the opposite direction, n=322) and Conv.-associated (convergent loop sharing one anchor with a nonconvergent loop in the opposite direction, n=496). Boxplots defined as in Fig. 1c. Two-sided BenjaminiHochberg-corrected MannWhitney U-test P=6.2106. d, Aggregated loop signal for categories in c. Arrows represent CBS orientation. e, Pax3 WISH in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Note the positive correlation between expanded Pax3 misexpression and increased number of deleted CBSs. Scale bar, 250m. f, Pax3 qPCR analysis in E11.5 limbs from CBS mutants. Bars represent mean and dots individual replicates. Values were normalized against DelBs mutant (Ct). Note the positive correlation of Pax3 misexpression with the increase in deleted CBSs (Pearson correlation significantly>0; ***P0,001). g, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. h, Insulation score values. Dots represent the local minima of the insulation score at the EP boundary for each mutant. i, Virtual 4C profiles for the region in g (viewpoint in Pax3). The gray rectangle highlights Epha4 enhancers. Note increased interactions between the Pax3 promoter and the Epha4 enhancers in R-all compared with DelBs.

We searched for further biological indications of this mechanism by analyzing ultra-high-resolution Hi-C datasets26. First, we identified loop anchors and classified them according to the orientation of their CBS motif and associated loops. Loop anchors were split into convergent-only (only CBSs oriented in the same direction as their anchored loops), nonconvergent (anchor loops in a direction for which they lack a directional CBS) and no-CTCF (no CBS). While most loop anchors belong to the convergent-only category14,36, 7.6% of them were classified as nonconvergent. Then, we explored whether these nonconvergent loops could be explained by the nonconvergent anchor simultaneously establishing a convergent loop in the opposite direction (Fig. 3a). We calculated the frequency of anchors involved in bidirectional loops for each category and discovered that, while only 5% of convergent-only or no-CTCF anchors participate in bidirectional loops, this percentage increases significantly up to 45% for nonconvergent anchors (Fig. 3b; chi-squared test, P<10225). To gain further insights into the mechanisms that establish convergent/nonconvergent loop pairs, we calculated the strength of each corresponding paired loop22. We observed that the convergent loops linked to a nonconvergent loop are significantly stronger than their nonconvergent counterparts (Fig. 3c,d; MannWhitney U-test, P=6106). Next, we explore if convergent loops paired to nonconvergent loops are particularly strong in comparison with other types of convergent loops. This analysis revealed that the strength of these convergent loops is similar to other unpaired convergent loops across the genome (Extended Data Fig. 6; single-sided convergent category). However, paired convergent/nonconvergent loops appear to be mechanistically different from unpaired loops, as they are more often associated with TAD corners (Extended Data Fig. 6c; chi-squared test, P<3.5106) and therefore connect anchor points that are located farther away in the linear genome (Extended Data Fig. 6d; MannWhitney U-test, P<4.8108). A comparison against pairs of convergent/convergent loops, which are similarly associated with TAD corners (Extended Data Fig. 6b; category double-sided convergent), revealed that the convergent loops in convergent/nonconvergent pairs are on average stronger (MannWhitney U-test, P=7105). This type of convergent/nonconvergent loops can be observed at relevant developmental loci, such as the Osr1, Ebf1 and Has2 loci (Extended Data Fig. 7). Overall, our analyses suggest that a considerable number of nonconvergent loops could be mechanistically explained by the presence of a stronger and convergent chromatin loop in the opposite orientation and anchored by the same CBS.

To validate these findings in vivo, we sequentially retargeted our R1 mESCs to create a mutant that only retains the forward F1 and F2 sites, which have strong functionality (Fig. 2a,b). During the process, we obtained intermediate mutants with double (R1+R3) and triple CBS deletion (R1+R3+R4), as well as the intended quadruple knockout lacking all reverse CBSs (R-all). WISH revealed an expanded Pax3 expression pattern towards the posterior limb region, an effect that increases with the number of deleted CBSs (Fig. 3e). Expression analyses by qPCR confirmed a significant increasing trend in Pax3 misexpression levels across mutants (Fig. 3f; Pearson correlation>0, P2107). These results demonstrate again that R2, R3 and R4 are functionally redundant sites, despite the absence of measurable effects upon individual deletions (Fig. 1b). However, we noted that Pax3 levels were only moderately increased (threefold) compared with the expression in mutants retaining only-reverse CBSs (ninefold, F-all). Importantly, R-all mutants retain two intact CBSs in the forward orientation, while up to four CBSs are still present in F-all mutants, suggesting that these two forward CBSs (F1 and F2) grant most of the insulator activity of the EP boundary. These experiments indicate that the functional characteristics of specific CBSs can outweigh other predictive parameters of boundary function such as the total number of sites.

As expected, cHi-C maps from R-all mutant limbs revealed a clear partition between the Epha4 and Pax3 TADs (Fig. 3g), consistent with the reduced Pax3 misexpression. Boundary scores at the EP boundary were also only moderately reduced (Fig. 3h), in comparison with the broader effects of the F-all mutant (Fig. 2d). Accordingly, intra-TAD interactions modestly decreased while inter-TAD interactions increased, as also observed in virtual 4C profiles (Fig. 3i). Despite the multiple deletions, the telomeric chromatin loops remained unaffected and anchored by the F1 and F2 sites, both occupied by RAD21 (Fig. 3g and Extended Data Figs. 4 and 5). However, we noticed the persistence of centromeric chromatin loops anchored by the F1 and F2 sites, despite their nonconvergent forward orientation. A higher contact intensity is observed at F1, which would be the first CBS encountered by cohesin complexes sliding from the centromeric side (Extended Data Figs. 4 and 5).

Finally, we investigated if the formation of nonconvergent loops might be associated with the accumulation of cohesin complexes over a limited number of CBSs. We generated a mutant that only retains the R3 CBS (R3-only), which is prominently bound by CTCF (Fig. 1b). We hypothesized that, in the absence of others, this CBS may accumulate the cohesin and form a nonconvergent loop. However, although R3 was the only site able to stall cohesin in this background (Extended Data Fig. 4), cHi-C maps revealed a single convergent loop towards the centromeric side (Extended Data Fig. 8). This loop displays a weak insulator function, denoted by a decreased boundary score, an Epha4 and Pax3 TAD fusion and prominent Pax3 misexpression. Therefore, our results in transgenic mice support our findings at the genome-wide level (Fig. 3ac), demonstrating that specific CBSs can create chromatin loops independently of their motif orientation, seemingly through loop interference.

Previous studies identified divergent CBS clusters as a signature of TAD boundaries, suggesting a role on insulation13,31. While our analysis on mutants with reverse-only CBS orientation (F-all) showed a severe impairment of boundary function (Fig. 2c), this was not the case for R-all mutants, which retain CBSs only in the forward orientation (Fig. 3f). Indeed, the levels of Pax3 misexpression evidenced that insulation is more preserved in R-all than in R1+F2 mutants, which still conserve a divergent CBS signature (Fig. 2c).

This prompted us to explore the relation between CBS composition at boundaries and insulation strength. We examined available Hi-C datasets, classifying boundary regions according to different parameters of CBS composition (that is, number and orientation) and calculating boundary scores (Fig. 4a). Our analysis revealed that, for the same CBS number, boundaries with divergent signatures generally display more insulation than their nondivergent counterparts. However, up to 6% of nondivergent boundaries display scores above 1.0, a value associated with robust functional insulation (Fig. 1c). Manual inspection at specific loci showed that nondivergent boundaries with strong boundary scores present clear TAD partition and no evidence of coregulation for genes located at either side (Extended Data Fig. 9). These results suggest that a divergent signature is not strictly required to form strong functional boundaries.

a, Relation between BSs and the number of CBSs for divergent and nondivergent boundaries in mESC Hi-C data26. Boxplots defined as in Fig. 1c. b, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Light-gray rectangle marks inverted region. Note similar Pax3 misexpression pattern between F-all-Inv and F-all mutants. Scale bar, 500m. c, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (two-sided t-test P value). d, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data mapped on custom genome containing the DelBs deletion and the inverted EP boundary (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. e, Insulation score values. Lines represent mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. f, Virtual 4C profiles for the genomic region displayed in d (viewpoint in Pax3). Light-gray rectangle highlights Epha4 enhancer region. Note similar interaction profile between F-all-Inv (yellow) and F-all mutants (orange).

Next, we explored if the genomic contexts might explain the prominent insulation differences between only-reverse (F-all) or only-forward (R-all) mutants. To evaluate this, we generated a mutant with a homozygous inversion of the boundary region, on the F-all background (F-all-Inv) (Fig. 4b and Supplementary Fig. 5).

WISH and qPCR experiments showed that Pax3 expression is almost indistinguishable from the F-all mutants, both spatially and at the quantitative level (Fig. 4b,c). Moreover, cHi-C maps from F-all-Inv mutants revealed a similar fusion of the Epha4 and Pax3 TADs (Fig. 4d). However, subtraction maps showed a redirection of chromatin loops, which now interact mainly with the telomeric Pax3 boundary instead of the centromeric Epha4 boundary. These ectopic loops are mainly anchored by the R1 site, which preserves its marked functionality. Despite these local differences, boundary scores and virtual 4C profiles remained comparable between F-all-Inv and F-all mutants (Fig. 4e,f). These results suggest that the orientation of entire boundary regions, as well as the differences in the surrounding genomic context, play a minor role in insulator function.

To determine to what extent CTCF binding contributes to the EP boundary function, we generated a sextuple knockout with all CBSs deleted (ALL). WISH revealed a further expansion of Pax3 misexpression, covering the distal limb entirely. This expanded expression mirrors that of DelB mutants, in which the entire boundary region is deleted (Fig. 5a). Expression analyses revealed that Pax3 misexpression in ALL mutants exceeds the combined sum of expression from R-all and F-all mutants (Fig. 5b), again indicating the cooperative and redundant CBS action. Intriguingly, Pax3 misexpression in the R3-only background was comparable to ALL, suggesting that a functionally weak CBS is not sufficient to hinder enhancerpromoter communication (Extended Data Fig. 8). Nevertheless, ALL mutants only reach 65% of the Pax3 misexpression observed in DelB mutants (Fig. 5b), which may be attributed to the 150-kb inter-CBS region that differentiates both mutants.

a, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs and the gray rectangle represents the deleted region. Note the similarities in expression pattern between mutants. Scale bar, 250m. b, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutants (Ct) (*two-sided t-test P0.05, ALL versus DelB: 0.03). c, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data mapped on custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs (left) and DelB (right). d, Insulation score values. Lines represent mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. e, Virtual 4C profiles for the genomic region displayed in c (viewpoint in Pax3). Light-gray rectangle highlights Epha4 enhancer region.

To investigate the reduced Pax3 misexpression in ALL, compared with DelB mutants, we performed cHi-C experiments (Fig. 5c). These experiments revealed a prominent Epha4 and Pax3 TAD fusion, with increased intensity of the meta-TAD loop (Extended Data Fig. 4). This results from the severe disruption of the EP boundary, denoted by a reduced boundary score (Fig. 5d) and the complete absence of RAD21 binding or anchored loops (Extended Data Figs. 4 and 5). In fact, the interaction profile at the EP boundary is not different from other internal locations of the Epha4 TAD (Fig. 5c). Of note, higher insulation is observed in R3-only compared with ALL, despite the comparable Pax3 misexpression between both genetic backgrounds (Extended Data Fig. 8). However, virtual 4C profiles from ALL and R3-only mutants confirmed a similar interaction between Epha4 enhancers and Pax3 (Fig. 5e and Extended Data Fig. 8). These enhancergene interactions were reduced in comparison with DelB, in which Pax3 misexpression is more prominent (Fig. 5e and Extended Data Fig. 8). ChIPseq datasets for epigenetic marks did not reveal additional regions with regulatory potential within the 150-kb region (Extended Data Fig. 3), indicating that the enhanced Pax3 misexpression in DelB mutants is unlikely caused by the deletion of regulatory elements. Taken together, these results suggest that enhancerpromoter distances might influence gene expression levels.

PAX3 misexpression during limb development can cause shortening of thumb and index finger (brachydactyly), in human patients and mouse models10. Therefore, our mutant collection provides an opportunity to study how boundary insulation strengths translate into developmental phenotypes.

We obtained mutant E17.5 fetuses and performed skeletal stainings, measuring relative digit length as a proxy for the phenotype (Fig. 6a,b). First, we analyzed R1 mutants, which displayed moderate Pax3 misexpression in the anterior distal limb (Fig. 1f). Finger length ratios revealed that R1 limbs develop normally, demonstrating that the detrimental effects of Pax3 misexpression can be partially buffered.

a, Skeletal staining of forelimbs from E17.5 mutant and control fetuses. White arrowheads indicate reduced index finger lengths. Black bracket shows the region of the finger measured for the quantification. Finger length correlates negatively with increased Pax3 misexpression. Scale bar, 500m. b, Index lengths relative to ring finger lengths in E17.5 mouse forelimbs. Bars represent the mean and white dots represent individual replicates. Values were normalized on control (CTRL) animals (two-sided t-test **P0.01; two-sided t-test ***P0.001; R1+F2 versus CTRL: 0.007; F-all versus CTRL: 0.0002). c, Correlation between the number of remaining CBSs at the EP boundary and the levels of Pax3 expression in the different mutants described in this study. Pearson regression lines are shown together with R2 values, both for the whole collection of mutants (black) and discarding combined CBS deletions involving CBSs with forward orientation (turquoise). d, Correlation and R2 between BSs and the brachydactyly phenotype penetrance measured as the index to ring finger length ratio for controls, R1+F2 and F-all mutants. The color of the dots represents the level of Pax3 limb misexpression as measured by qPCR. e, Model for boundary insulation as a quantitative modulator of gene expression and developmental phenotypes. Left, a strong boundary (B) efficiently insulates gene A from the enhancers located in the adjacent TAD (E). The boundary shows a cluster of CBSs with different orientations represented with arrowheads. The colored arrow represents a CBS with prominent contribution to boundary function. Middle, the absence of specific CBSs results in a weakened boundary, moderate gene misexpression (limb, indicated in yellow) and mild phenotypes (reduced digits, indicated in red and pointed out by white arrowhead). Right, the absence of the boundary causes a fusion of TADs, strong gene misexpression and strong phenotypes.

In contrast, R1+F2 mutants displayed a moderate reduction of index digit length (Fig. 6a,b), consistent with their increased Pax3 misexpression (Fig. 2b). This demonstrates that weakened boundaries can be permissive to functional interactions between TADs, resulting in altered transcriptional patterns and phenotypes. Importantly, the phenotypes of R1+F2 mutants occur despite an observable partition between Epha4 and Pax3 TADs and across a boundary region displaying high boundary scores (Fig. 2c,d; boundary score=0.8). Analyses on ultra-high-resolution Hi-C datasets26 revealed that many boundary scores fall within the ranges described in our mutant collection (Extended Data Fig. 10). Of note, 40% of boundaries display scores lower than 0.8. According to our observations, those boundaries could be permeable for functional interactions across domains.

Finally, we analyzed the F-all mutants, in which the Epha4 and Pax3 TADs appear largely fused (Fig. 2c). This disruption of TAD organization led to a prominent reduction of digit length (Fig. 6a,b), consistent with the higher Pax3 misexpression (Fig. 2b). Overall, these results illustrate how boundary insulation strength can modulate gene expression and developmental phenotypes, by allowing permissive functional interactions between TADs.

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In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation - Nature.com

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This five-day conference will be held Aug. 1418 in person in Cambridge, Massachusetts, and online. It will be an international forum for discussion of the remarkable advances in cell and human protein biology revealed by ever-more-innovative and powerful mass spectrometric technologies. The conference will juxtapose sessions about methodological advances with sessions about the roles those advances play in solving problems and seizing opportunities to understand the composition, dynamics and function of cellular machinery in numerous biological contexts. In addition to celebrating these successes, the organizers also intend to articulate urgent, unmet needs and unsolved problems that will drive the field in the future. The registration deadline was July 1, but you have until July 12 to register to participate virtually.Learn more.

For Discover BMB, the ASBMB's annual meeting in March in Seattle, we're seeking two types of proposals:

The American Physiological Society is hosting a free webinar that will cover polycystic ovary syndrome, an endocrine disorder associated with modestly elevated androgens, and hormone therapy for transmen, which elevates androgens greatly to achieve levels similar to those in cisgender men. The event announcement says: "The role that these two different concentrations play in cardiovascular physiology and pathophysiology remains unclear. Gaps and opportunities in basic research and clinical practice will be highlighted." The speaker will be Licy Yanes Cardozo, a physician-scientist at the University of Mississippi Medical Center. Learn more and register.

In May, the Howard Hughes Medical Institute launched a roughly $1.5 billion program to "help build a scientific workforce that more fully reflects our increasingly diverse country." The Freeman Hrabowski Scholars Program will fund 30 scholars every other year, and each appointment can last up to 10 years. That represents up to $8.6 million in total support per scholar. HHMI is accepting applications from researchers "who are strongly committed to advancing diversity, equity, and inclusion in science." Learn more.

Save the date for the ASBMB Career Expo. This virtual event aims to highlight the diversity of career choices available to modern biomedical researchers. No matter your career stage, this expo will provide a plethora of career options for you to explore while simultaneously connecting you with knowledgeable professionals in these careers. Each 60-minute session will focus on a different career path and will feature breakout rooms with professionals in those paths. Attendees can choose to meet in a small group with a single professional for the entire session or move freely between breakout rooms to sample advice from multiple professionals. Sessions will feature the following five sectors: industry, government, science communication, science policy and other. The expo will be held from 11 a.m. to 5 p.m. Eastern on Nov. 2. Stay tuned for a link to register!

The Journal of Science Policy & Governanceand the National Science Policy Network issued a call for papersfor an issue containingpolicy ideas from the next generation of scientists. The submission deadline is Nov. 6. Theyencourage submissions "that highlight policy opportunities and audiences related to the 2022 U.S. midterm elections at the local, stateor national level as well as related foreign policy issues."Read the press release.

The ASBMB provides members with a virtual platform to share scientific research and accomplishments and to discuss emerging topics and technologies with the BMB community.

The ASBMB will manage the technical aspects, market the event to tens of thousands of contacts and present the digital event live to a remote audience. Additional tools such as polling, Q&A, breakout rooms and post event Twitter chats may be used to facilitate maximum engagement.

Seminars are typically one to two hours long. A workshop or conference might be longer and even span several days.

Prospective organizers may submit proposals at any time. Decisions are usually made within four to six weeks.

Propose an event.

If you are a graduate student, postdoc or early-career investigator interested in hosting a #LipidTakeover, fill out this application. You can spend a day tweeting from the Journal of Lipid Research's account (@JLipidRes) about your favorite lipids and your work.

The International Union of Biochemistry and Molecular Biology is offering $500 to graduate students and postdocs displaced from their labs as a result of natural disaster, war or "other events beyond their control that interrupt their training." The money is for travel and settling in. Learn more and spread the word to those who could use assistance.

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Calendar of events, awards and opportunities - ASBMB Today

Engineering | Health and medicine | Science | UW News blog

July 15, 2022

Another beautiful day on the University of Washingtons Seattle campus.University of Washington

Seven professors at the University of Washington are among 25 new members of the Washington State Academy of Sciences, according to a July 15 announcement. Joining the academy is a recognition of their outstanding record of scientific and technical achievement, and their willingness to work on behalf of the Academy to bring the best available science to bear on issues within the State of Washington.

Twenty of the incoming members for 2022 were selected by current WSAS members, while the other five were chosen by virtue of recently joining one of the National Academies.

UW faculty selected by current Academy members are:

In addition, Dr. Jay Shendure, UW professor of genome sciences, investigator with the Howard Hughes Medical Institute and faculty member in the Molecular Engineering & Sciences Institute, was selected by virtue of his election to the National Academy of Sciences for pioneering a variety of genome sequencing and analysis methods, including exome sequencing and its earliest applications to gene discovery for Mendelian disorders and autism; cell-free DNA diagnostics for cancer and reproductive medicine; massively parallel reporter assays; saturation genome editing; whole organism lineage tracing; and massively parallel molecular profiling of single cells.

New members to the Washington State Academy of Sciences will be formally inducted in September.

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Seven UW faculty members elected to the Washington State Academy of Sciences - University of Washington

A recent study led by the University of Wisconsin Madison has found that some species of copepods such as Eurytemora affinis tiny crustaceans measuring about a millimeter in length and roaming coastal waters of oceans and estuaries around the world in massive numbers can evolve fast enough to survive in the face of rapid climate change.

This is a dominant coastal species, serving as very abundant and highly nutritious fish food, said study senior author Carol Eunmi Lee, a professor of Integrative Biology at UW Madison. But theyre vulnerable to climate change.

Since ocean salinity is dropping rapidly as ice melts and precipitation patterns change, this saltwater species that evolved over the ages in waters high in salinity, now needs to adapt to much fresher water in their environment.

In order to study how copepods respond to drops in salinity, the scientists kept a population of Eurytemora affinisfrom the Baltic Sea in their laboratory and observed them over a few generations. After splitting the copepods into 14 groups of a few thousands each, they placed four of these groups in environments similar to the Baltic, while exposing the other ten groups to declining salinity levels that simulated the type of pressure caused by climate change. For a total of ten generations, these groups had their water gradually reduced to lower salinity levels.

To track evolutionary changes across the genomes of the tiny crustaceans, the researchers sequenced the genome of each line of copepods at the beginning of the experiment, as well as after six and ten generations.

The analysis revealed that the strongest signals of natural selection where changes were largest and more frequent across the groups exposed to low salinity levels were in areas of the genome that are important in regulating ions, such as sodium transporters.

In saltwater, there are a lot of ions, like sodium, that are essential for survival. But when you get to freshwater, these ions are precious, Professor Lee explained. So, the copepods need to suck them up from the environment and hang on to them, and the ability to do that relies on these ion transporters that we found undergoing natural selection.

At the end of the experiment, the copepods with certain genetic combinations of the ion transporter were more likely to survive, even as the salinity of their water decreased. According to the researchers, the gene variants found in the copepods that managed to survive the salinity decline in the laboratory are also common among copepods living in the fresher regions of the Baltic Sea.

This copepod gives us an idea of what it takes, an idea of what conditions are needed, that enable a population to evolve rapidly in response to climate change. It also shows how important evolution is for understanding our changing planet and how or even whether populations and ecosystems will survive, Professor Lee concluded.

The study is published in the journal Nature Communications.

By Andrei Ionescu, Earth.com Staff Writer

Continued here:
Tiny crustaceans have what it takes to survive climate change - Earth.com

We have long been baffled as to why men live around five years less than women, on average. But now a new study suggests that, beyond the age of 60, the main culprit is a genetic defect: the loss of the Y chromosome, which determines sex at birth.

Its clear that men are more fragile, the question is why, explains Lars Forsberg, a researcher at Uppsala University in Sweden.

For decades it was thought that the male Y chromosomes only function was to generate sperm that determine the sex of a newborn. A boy carries one X chromosome from the mother and one Y from the father, while a girl carries two Xs, one from each parent.

In 1963, a team of scientists discovered that as men age, their blood cells lose the Y chromosome due to a copying error that happens when the mother cell divides to produce a daughter cell. In 2014, Forsberg analyzed the life expectancy of older men based on whether their blood cells had lost the Y chromosome, a mutation called mLOY. The effect recorded was mindblowing, the researcher recalls.

Men with fewer Y chromosomes had a higher risk of cancer and lived five and a half years less than those who retained this part of the genome. Three years later, Forsberg discovered that this mutation makes getting Alzheimers three times as likely. What is most worrying is the enormous prevalence of this defect. Twenty percent of men over the age of 60 have the mutation. The rate rises to 40% in those over 70 and 57% in those over 90, according to Forsbergs previous studies. It is undoubtedly the most common mutation in humans, he says.

Until now, nobody knew whether the gradual disappearance of the Y chromosome in the blood played a pivotal role in diseases associated with aging. In a study just published in the journal Science, Forsberg and scientists from Japan and the US demonstrate for the first time that this mutation increases the risk of heart problems, immune system failure and premature death.

The researchers have created the first animal model without a Y chromosome in their blood stem cells: namely, mice modified with the gene-editing tool CRISPR. The study showed that these rodents develop scarring of the heart in the form of fibrosis, one of the most common cardiovascular ailments in humans, and die earlier than normal mice. The authors then analyzed the life expectancy recorded in nearly 15,700 patients with cardiovascular disease whose data are stored in the UK public biobank. The analysis shows that loss of the Y chromosome in the blood is associated with a 30% increased risk of dying from cardiovascular disease.

This genetic factor can explain more than 75% of the difference in life expectancy between men and women over the age of 60, explains biochemist Kenneth Walsh, a researcher at the University of Virginia in the US and co-author of the study. In other words, this mutation would explain four of the five years lower life expectancy in men. Walshs estimate links to a previous study in which men with a high mLOY load live about four years less than those without it.

It is well known that men die earlier than women because they smoke and drink more and are more prone to recklessness. But, beyond the age of 60, genetics becomes the main culprit in the deterioration of their health: It seems as if men age earlier than women, Walsh points out.

The study reveals the molecular keys to the damage associated with the mLOY mutation. Within the large group of blood cells can be found the immune systems white blood cells responsible for defending the body against viruses and other pathogens. The loss of the Y chromosome triggers aberrant behavior in macrophages, a type of white blood cell, causing them to scar heart tissue, which in turn increases the risk of heart failure. Researchers have shown that the damage can be reversed if they give mice pirfenidone, a drug approved to treat humans with idiopathic pulmonary fibrosis, a condition in which the lungs become scarred and breathing becomes increasingly difficult.

There are three factors that increase the risk of Y chromosome loss. The first is the inevitable ageing process. The longer one lives, the more cell divisions occur in the body and the greater the likelihood of mutations occurring in the genome copying process. The second is smoking. Smoking causes you to lose the Y chromosome in your blood at an accelerated rate; if you stop smoking, healthy cells once again become the majority, says Walsh. But the third is also inevitable: other inherited genetic mutations can increase the gradual loss of the Y chromosome in the blood by a factor of five, explains Forsberg.

Both Forsberg and Walsh believe that this study opens up an enormous field of research. Still to be studied is whether men with this mutation also have cardiac fibrosis and whether this is behind their heart attacks and other cardiac ailments. We also need to better understand why losing the Y chromosome damages health. For now, we have shown that the Y chromosome is not just there for reproduction, but is is also important for our health, says Forsberg. The next step is to identify which genes are responsible for the phenomenon.

The loss of this chromosome has been detected in all organs and tissues of the body and at all ages, although it is more evident after 60. It is abundant in the blood because this is a tissue that produces millions of new cells every day from blood stem cells. Healthy stem cells produce healthy daughter cells and mutated ones produce daughter cells with mLOY.

A previous study showed that this mutation of the Y chromosome disrupts the function of up to 500 genes located elsewhere in the genome. It has also been shown to damage lymphocytes and natural killer cells, evident in men with prostate cancer and Alzheimers disease, respectively.

There are hardly any tests for mLOY at present. But Forsberg and his colleagues have designed a PCR test that measures the level of this mutation in the blood and could serve to determine which levels of this mutation are harmful to health. Right now, we see people in their 80s with 80% of their blood cells mutated, but we dont know what impact this has on their health, says Walsh.

Another unanswered question is why men lose the genetic mark of the male with age. The evolutionary logic, argue the authors of the paper, is that men are biologically designed to have offspring as soon as possible and to live 40 to 50 years at most. The spectacular increase in life expectancy in the last century has meant that men and women live to an advanced age 80 and 86 years in Spain, respectively which makes the effect of these mutations more evident. Another fact which possibly has some bearing on the issue: the vast majority of people who reach 100 are women.

To transform all these discoveries into treatments, we first need to better understand this phenomenon, says Forsberg. We men are not designed to live forever, but perhaps we can increase our life expectancy by a few more years.

Biochemist Jos Javier Fuster, who studies pathological mutations in blood cells at the National Center for Cardiovascular Research, stresses the importance of the work. Until now it was not clear whether the loss of Y was the cause of cancer, Alzheimers disease and heart failure, he explains. This is the first demonstration in animals that it has a causal role. The human Y chromosome is different from the mouse chromosome, so the priority now is to accumulate more data in humans. This is a great first step in understanding this new mechanism behind aging-linked diseases, he adds.

The cells of the human body group their DNA into 23 pairs of chromosomes that pair up one by one when a cell copies its genome to generate a daughter cell. The Y is the only one that does not have a symmetrical partner to pair up with: instead, it does so with an X chromosome; and the entire Y chromosome is often lost, explains Luis Alberto Prez Jurado from Pompeu Fabra University in Barcelona. For now, six genes have been identified within the Y chromosome that would be responsible for an impact on health, he says. All of them are related to the proper functioning of the immune system. In part, this would also explain the greater vulnerability of males to viral infections, including Covid-19.

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mLOY: The genetic defect that explains why men have shorter lives than woman - EL PAS USA

When Josefin Stiller was growing up in Berlin, she loved reading about Greek gods in an encyclopedia of mythology. She often lost track of their relationships, howevertheir feuds, trysts, and betrayalsas she flipped among the entries. Frustrated, she wrote each name on a card and started to arrange children beneath parents on a desk in her bedroom. As lineages became clear, so did family dramas. Sons killed fathers; uncles kidnapped nieces; siblings fell in love. I wonder if this experience of reconstructing a family tree primed me to appreciate trees and the powerful insights they hold, Stiller told me in a recent e-mail.

Years later, as a graduate student in biology, Stiller worked on an evolutionary tree for seahorses and their relatives, using DNA to understand the ancestry of different species. Then, in 2017, she moved to the University of Copenhagen and joined B10K, a scientific collaboration that aims to sequence the genome of every bird speciesmore than ten thousand in alland to reveal their connections in a comprehensive tree. The amount of data and computing power required for this mission is almost unfathomable, but the final product should be as simple in principle as the diagram Stiller had assembled as a child. Everything in biology has a history, and we can show this history as a bifurcating tree, she said.

Birds are the most diverse vertebrates on land, and they have always been central to ideas about the natural world. In 1837, a taxonomist in London told Charles Darwin that the finches he had shot and carelessly lumped together in the Galpagos Islands were, in fact, many different species. Darwin wondered whether the finches might have shared a common ancestor from mainland South Americawhether all of life might have evolved through a process of descent with modificationand he drew a rudimentary tree in his private notebook, beneath the words I think. The tree showed how a single ancestral population could branch into many species, each with its own evolutionary path. On the Origin of Species, published twenty-two years later, includes only one diagram: an evolutionary tree. The tree of life became for biology what the periodic table was for chemistryboth a foundation and an emblem for the field. Thetime will come I believe, though I shall not live to see it, when we shall have fairly true genealogical treesofeach great kingdomofnature, Darwin wrote to a friend.

The rise of genome sequencing, at the turn of the twenty-first century, seemed to bring Darwins dream within reach. It is now realistic to conceive of reconstructing the entire Tree of Lifeeventually to include all of the living and extinct species, Joel Cracraft, the curator of birds at the American Museum of Natural History, wrote, in 2004. The naturalist E. O. Wilson predicted that such a tree could unify biology. Its value to such fields as agriculture, conservation, and medicine would be incalculable; evolutionary trees have already deepened our understanding of SARS-CoV-2, the virus that causes COVID-19. By mapping a major branch on the tree of life, B10K aims to light the way.

When Stiller joined the project, her colleagues were combing through museums and laboratories to sample three hundred and sixty-three bird species, chosen carefully to represent the diversity of living birds. With help from four supercomputers in three different countries, they began to compare each birds DNA to figure out how they were related. I think there was always this idea that, once we sequence full genomes, we will be able to solve it, Stiller told me. But, early in the process, she encountered an evolutionary enigma called Opisthocomus hoazin. I was completely amazed by this bird, she said.

Hoatzins, which live along oxbow lakes in tropical South America, have blood-red eyes, blue cheeks, and crests of spiky auburn feathers. Their chicks have primitive claws on their tiny wings and respond to danger by plunging into water and then clawing their way back to their nestsa trait that inspired some ornithologists to link them to dinosaurs. Other taxonomists argued that the hoatzin is closely related to pheasants, cuckoos, pigeons, and a group of African birds called turacos. Alejandro Grajal, the director of Seattles Woodland Park Zoo, said that the bird looks like a punk-rock chicken, and smells like manure because it digests leaves through bacterial fermentation, similar to a cow.

DNA research has not solved the mysteries of the hoatzin; it has deepened them. One 2014 analysis suggested that the birds closest living relatives are cranes and shorebirds such as gulls and plovers. Another, in 2020, concluded that this clumsy flier is a sister species to a group that includes tiny, hovering hummingbirds and high-speed swifts. Frankly, there is no one in the world who knows what hoatzins are, Cracraft, who is now a member of B10K, said. The hoatzin may be more than a missing piece of the evolutionary puzzle. It may be a sphinx with a riddle that many biologists are reluctant to consider: What if the pattern of evolution is not actually a tree?

Fossils that resemble hoatzins have been found in Europe and Africa, but today the birds can be found only in the river basins of the Amazon and Orinoco of South America. I live in Germany, so I visited them in Berlins Museum of Natural History, where cabinets are filled with thousands of stuffed birds. Sylke Frahnert, the bird curator, kept two taxidermy hoatzins on a shelf near the cuckoos and turacos, which seems as good a place as any. Over the years, there have been so many conflicting trees of birds, she told me. You would have been crazy to change the collection with every one. One of the museums hoatzins was shot in Brazil more than two centuries ago, and the years have drained the color from its face. I had heard that even the specimens smell like manure, but Frahnert warned me not to sniff them, since birds were once preserved with arsenic.

In the eighteenth century, natural-history museums started using anatomical similarities to classify plants and animals into increasingly specific categories: class, order, family, genus, species. Darwin realized that species share traits because their ancestors were one and the same. Fish, amphibians, reptiles, birds, and mammals all have spines, but not because God had given them to each creature separately; rather, the spine suggested a common parent living long ago. The construction of evolutionary trees was dubbed phylogeny, literally meaning the generation of species, by the zoologist Ernst Haeckel. The more traits two species shared, the theory went, the more recently they had shared a common ancestor. Human beings and other great apes evolved from a common ancestor millions of years ago, but even human beings and bacteria have a common ancestorthe first known living organisms, which date to three and a half billion years ago.

Hoatzinsin some respects the most aberrant of birds, according to one Victorian ornithologistwere a problem from the beginning. Early European naturalists described them as pheasants, and the first major tree for birds, published in 1888 by Max Frbringer, placed them on the fowl branch. But, by the early nineteen-hundreds, some scientists were comparing hoatzins and cuckoos on the basis of traits such as jaws and feathers, and others were noting similarities between hoatzins and turacos, pigeons, barn owls, and rails. Even the hoatzins parasites defied classification: they hosted feather lice found on no other birds.

One crucial problem in phylogeny was convergent evolution. Sometimes natural selection nudges two organisms toward the same trait. Birds and bats independently evolved the ability to fly. Swifts and swallows each evolved into aerodynamic insectivores with nearly identical silhouettes, but traits such as their vocal organs and foot bones reveal that they are only distantly related. Because taxonomists often disagreed about things such as how to distinguish common ancestry from convergent evolution, the literature grew thick with conflicting trees, to the point that some twentieth-century biologists seemed ready to give up. The construction of phylogenetic trees has opened the door to a wave of uninhibited speculation, one wrote in 1959. Science ends where comparative morphology, comparative physiology, comparative ethology have failed us.

Phylogeny made a comeback in the seventies and eighties, after the German entomologist Willi Hennig developed more rigorous criteria for identifying common ancestry and drawing evolutionary trees. These innovations laid a foundation for a new wave of research that did not rely solely on physical specimens but, rather, on the emerging science of DNA. Organisms are related to one another by the degree to which they share genetic information, two ornithologists wrote in the early nineties, adding that genetics could reveal a different view of the process of evolution and its effects. The typical bird genome is a string of more than a billion base pairs that mutate randomly over time. Scientists can compare the same parts of the genome across multiple species to estimate their evolutionary closeness. Typically, species that share mutations have a more recent common ancestor, and species that do not are more distantly related.

Early sequencing was expensive and tedious, but, by the beginning of the twenty-first century, a signal was emerging from the noise. The journal Nature published an article about the promise of a single unified tree of life. But its author also identified a complication: each genome contains many different genes, and each one could generate a different evolutionary tree.

In 2001, a paper in the Proceedings of the Royal Society identified a pair of bird siblings as unlikely as Arnold Schwarzenegger and Danny DeVito: the flamingos closest relative was a little diving bird called a grebe. That was probably the single most astounding result that anybodys ever gotten, Peter Houde, an avian biologist from New Mexico State University, told me. Ornithologists had always reasoned that grebes were closely related to short-legged loons, whereas tall wading birds such as flamingos, storks, and herons probably had a long-legged common ancestor.

That was the first domino to fall. In 2008, Science published a new avian tree based on DNA. Research led by Shannon Hackett, Rebecca Kimball, and Sushma Reddy, scientists affiliated with the Field Museum and the University of Florida, examined nineteen parts of the genomes of a hundred and sixty-nine avian species. The root of their tree resembled trees based on physical specimens: large, flightless birds such as ostriches, emus, and kiwisknown collectively as ratiteswere first to diverge from all the others, followed by land fowl and waterfowl. The remaining ninety-five per cent of living birds, from parrots to penguins and pigeons, are known as modern birds and descended from a common ancestor, probably around the time that an asteroid hit the earth, sixty-six million years ago, and the dinosaurs went extinct. The youngest orderpasserines, which include all songbirdsbranched out into a staggering six thousand species in the span of tens of millions of years. The genetic tree for modern birds was decked with relationships that few, if any, taxonomists had guessed from anatomy; key groups such as parrots, owls, woodpeckers, vultures, and cranes shifted places.

Scientists had long assumed, for example, that daytime hunters such as hawks, eagles, and falcons all descended from a single bird of prey. But, in the genetic tree, hawks and eagles shared a branch with vultures, yet falcons turned out to be closer relatives of passerines and parrots. This meant that the peregrine falcon is more closely related to colorful macaws and tiny sparrows than to any hawk or eagle. The traditional explanation for flightlessness in ratitesthat a common ancestor diverged into ostriches, emus, rheas, cassowaries, and kiwis after the southern continents split apartalso collapsed. DNA showed that the ratites also included flying birds called tinamous, suggesting that the group evolved flightlessness at least three separate times. That study revolutionized our understanding of how the major groups of living birds are related to each other, Daniel J. Field, an avian paleontologist at the University of Cambridge, said. Bird-watching guides had to reorganize their contents to reflect the new relationships.

Excerpt from:
The Bizarre Bird Thats Breaking the Tree of Life - The New Yorker

NEWARK, Calif., July 12, 2022 /PRNewswire/ --Ultima Genomics, Inc. (the Company) has signed an agreement with Regeneron Pharmaceuticals, Inc. (Nasdaq: REGN) to further advance Ultima Genomics' sequencing architecture. Under the terms of the agreement, Regeneron will collaborate with Ultima on the development and testing of Ultima's second-generation sequencing platform, which will build upon the advances of the Company's first instrument, the UG100anticipated to launch in 2023. In conjunction with the agreement, Regeneron, who is currently part of the early access program for the UG100, will also become an investor in Ultima. The primary objective of the collaboration between Ultima and Regeneron is to enable affordable high-throughput sequencing for large-scale genomic analysis and to accelerate insights and discoveries that will profoundly impact life sciences research around the world.

"Regeneron and Ultima share a common goal of using science to improve human health," said John Overton, Ph.D., Vice President and Chief of Sequencing and Lab Operations at the Regeneron Genetics Center (RGC). "With more than 120 active research collaborations and one of the largest whole exome sequencing reference libraries in the world, we at the RGC are keenly interested in the development of technologies that streamline the drug discovery and development process. The high cost of next-generation sequencing constrains the production of genomic information a significant bottleneck for life sciences research. With this agreement, we hope to contribute to an affordable and scalable solution that enables the rapid advance of genomic sciences, and in turn, important medicines for patients in need."

"We founded Ultima Genomics with the mission to continuously drive the scale of genomic information," said Gilad Almogy, Ultima Genomics' founder and Chief Executive Officer."While we will soon be launching our first instrument platform, the UG100, we are already hard at work developing our second platform to provide even lower cost and greater scale. We are excited to collaborate with Regeneron on this project and look forward to providing tools with the ever-increasing capability to our customers. Throughout our development, we have relied on, and are grateful for, the support, trust, and relationship with collaborators such as Regeneron, all of which are among the leading sequencers in the world."

Over the last five years, Ultima Genomics has developed a fundamentally new sequencing architecture designed to scale beyond conventional approaches and enable sequencing at a fraction of the cost of other commercially available technologies. The new architecture includes completely different approaches to flow cell engineering, sequencing chemistry, and machine learning.Ultima is currently in an early access program for the UG100, its first high throughput NGS instrument using the new technology architecture. The second-generation instrument will further increase the scale of genomic data and reduce sequencing costs beyond the first instrument. The timing of development and commercial availability of the Company's second instrument is not yet disclosed.

About Ultima Genomics

Ultima Genomics is unleashing the power of genomics at scale. The Company's mission is to continuously drive the scale of genomic information to enable unprecedented advances in biology and improvements in human health. With humanity on the cusp of a biological revolution, there is a virtually endless need for more genomic information to address biology's complexity and dynamic change and a further need to challenge conventional next-generation sequencing technologies. Ultima's revolutionary new sequencing architecture drives down the costs of sequencing to help overcome the tradeoffs that scientists and clinicians are forced to make between the breadth, depth, and frequency with which they use genomic information. The new sequencing architecture was designed to scale far beyond conventional sequencing technologies, lower the cost of genomic information and catalyze the next phase of genomics in the 21st century. To learn more, visit http://www.ultimagenomics.com

Media inquiries: [emailprotected]

SOURCE Ultima Genomics

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Ultima Genomics signs development agreement with Regeneron aimed at driving the scale of genomic information for drug discovery and development - PR...

SkyQuest Technology Consulting Pvt. Ltd.

Global next generation sequencing market was valued at $10.28 billion in 2021, and it is expected to reach a value of $33.73 billion by 2028, at a CAGR of 18.50% over the forecast period (20222028).

Westford, USA, July 13, 2022 (GLOBE NEWSWIRE) -- Next generation sequencing (NGS) is a game-changing technology that is revolutionizing how we study and understand biology. NGS allows us to sequence vast amounts of DNA or RNA much faster and more cheaply than ever before, making it possible to generate unprecedented amounts of data about the genomes of organisms. The demand for NGS services in the global next generation sequencing market has been growing rapidly in recent years, as the technology has become more affordable and accessible. A wide variety of scientific disciplines are now using NGS, including human genomics, cancer research, microbiology, evolutionary biology, agriculture, and many others. The number of publications featuring NGS data has also been increasing rapidly in recent years.

One of the key factors driving the growth of the next generation sequencing market is the ongoing need for better methods for diagnosing and treating diseases. For example, NGS can be used to detect genetic variations that may be associated with disease risk. It can also be used to identify novel drug targets and develop personalized medicines. In addition, NGS is playing an increasingly important role in basic research as scientists strive to understand the complexities of genome function.

Next Generation Sequencing: A Magic Wand for Early Diagnosis of Cancer

The global prevalence of cancer is alarmingly high, with an estimated 17 million new cases and 10 million deaths in 2020 alone. The World Health Organization (WHO) predicts that these figures will rise to 27 million new cases and 16.3 million deaths by 2040 if current trends continue. Early detection of cancer through screening programs is one of the most effective ways to reduce the burden of this disease, as it can lead to earlier diagnosis and treatment, which can improve survival rates significantly. Apart from this, more than 400,000 children develop cancer every year.

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The next generation of sequencing technology is often heralded as a magic wand for the early diagnosis of cancer. This is because it has the potential to provide rapid and accurate identification of tumors at an early stage, when they are most treatable. Next generation sequencing (NGS) technology intervenes at the level of DNA, providing information on genetic variation within a tumor. This can be used to detect early-stage cancers that may not yet have developed symptoms or detectable changes at the cellular level. The Global next generation sequencing market is also gaining demand for monitoring the progression of a cancer, and to assess how well treatments are working.

There are several reasons why NGS is seen as such a powerful tool in cancer diagnosis and treatment. First, it is extremely sensitive and can detect very small amounts of DNA from a tumor. Second, it can rapidly generate large amounts of data, which can be used to identify even rare mutations that may be associated with cancer. Finally, NGS is relatively simple and inexpensive to perform, making it widely accessible across the global next generation sequencing market from almost all strata of society, especially in developed market like the US, the UK, and Germany, among others. Despite all these advantages, there are still some challenges associated with the use of NGS in cancer diagnosis and treatment. While NGS can identify DNA alterations that may be associated with cancer.

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Next Generation Sequencing is Becoming Popular among Parents to Check Inborn Errors

As more and more parents learn about the benefits of next generation sequencing (NGS), its popularity is growing as a tool to check for inborn errors. NGS can provide a more comprehensive picture of a child's health, allowing parents to catch potential problems early and get their children the treatment they need. While NGS is not yet perfect, it is becoming more reliable and affordable as technology improves. As more parents learn about its potential, next generation sequencing market is likely to become even more popular as a way to keep children healthy and catch problems early.

As the technology for DNA sequencing gets cheaper and more sophisticated, an increasing number of parents are opting to have their child's genome sequenced at birth. This is especially true for parents who have a family history of genetic diseases. Sequencing the genome of a newborn is becoming more popular as the technology gets cheaper and more sophisticated. Inborn errors of metabolism are a group of rare genetic disorders that can cause serious health problems. Many of these disorders in the global next generation sequencing market are difficult to diagnose, and they often go undetected until something goes wrong.

According to the National Institutes of Health, about 25% of all live births in the United States each year are affected by at least one hereditary disease. However, the prevalence of these disorders varies widely, depending on the specific condition. For example, conditions like cystic fibrosis and sickle cell disease are relatively common, while others like Huntingtons disease are much rarer.

There are many different types of hereditary diseases, and they can affect any organ or system in the body. Some of the more common conditions include heart defects, respiratory problems, mental retardation, metabolic disorders, and cancer. Many of these disorders are life-threatening or cause significant disability, so it is important to be aware if any family members who have been affected by them. As a result, people are increasingly focusing getting their genome sequenced to know if they are carrying any gene that can lead to cancer.

With advancement in the next generation sequencing market, parents can now choose to have their child's genome sequenced at birth. This gives them the ability to catch these disorders early and start treatment immediately. It also allows them to make informed decisions about their child's future health. Although the cost of sequencing a genome is still relatively high, it is dropping rapidly. And as the technology continues to improve, it is expected that more parents will choose to have their child's genome sequenced at birth.

Food Industry to Offer Lucrative Opportunity for Next Generation Sequencing Market as Safety Concern Rises

The application of next generation sequencing in food safety and quality is an area of great interest and promise. The use of next generation sequencing technologies has the potential to revolutionize how we monitor food safety and quality. By providing rapid and high-throughput sequence data, next generation sequencing can be used to detect pathogens and other microorganisms in food more quickly and accurately than ever before. Additionally, the application of next generation sequencing can help us to better understand the genetic basis of food spoilage and contamination.

Growing concern about food safety has led to stricter quality control measures in the food industry. In particular, there is a greater focus on ensuring that food products are free from contaminants and meet safety standards, which is offering a lucrative opportunity for the players active in the next generation sequencing market to make most out of it. To this end, food manufacturers are increasingly adopting quality management systems such as Hazard Analysis and Critical Control Points (HACCP). These systems help to identify and control potential hazards at all stages of the food production process, from raw materials to finished products.

There are many different applications of next generation sequencing market in food safety and quality. One example is the use of next generation sequencing for pathogen detection. Pathogens are a major cause of foodborne illness, and traditional methods for detecting them often have low sensitivity or take too long to provide results. However, next generation sequencing can be used to rapidly detect pathogens in food samples with high accuracy. This information can then be used to taking steps to prevent outbreaks before they occur. In addition to pathogen detection, another important application of next generation sequencing is monitoring antibiotic resistance in bacteria present in food items. Antibiotic resistance is a growing public health concern, as it makes infections harder to treat and increases the risk of potentially deadly superbugs infection.

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AI and Accelerated Computing to Bring Down the Cost of Next Generation Sequencing to Less Than $300

AI and accelerated computing are already enabling a wide range of new applications in genomics. One such application is next generation sequencing (NGS), which is used to determine the order of nucleotides in DNA. NGS is currently used for a variety of purposes, including diagnosing genetic diseases, determining ancestry, and predicting drug response in the global next generation sequencing market. The cost of NGS has fallen dramatically in recent years, from $5.6 billion per genome in 2001 to less than $1,000 today. However, there is still room for further improvement.

AI and accelerated computing can help bring down the cost of NGS by making it more efficient. For example, AI can be used to streamline the process of sequence alignment, which is often the most time-consuming and computationally intensive step in NGS. In addition, accelerated computing can be used to speed up other parts of the NGS process, such as variant calling and read mapping. The combination of AI and accelerated computing has the potential to reduce the cost of NGS even further, to less than $300 per genome. This would make it affordable for many more people to access this important technology.

Top 10 Biologics Companies in the US Raised Over 1.65 billion in 2021, but Ultima Genomics took the Larger Piece of Pie

These companies in the global next generation sequencing market are working on developing new treatments and cures for a variety of diseases and conditions. Some of the diseases that they are targeting include cancer, Alzheimers disease, Parkinsons disease, and multiple sclerosis. The amount of money that these companies have raised shows how important it is to find new treatments for these diseases. The hope is that with this additional funding, these companies will be able to make even more progress in developing new therapies and cures. In May 2022, the company managed to raise $600 million through private equity. The company is planning to use this funding to bring down the NGS to $100 in the years to come. Apart from this, Kriya Therapeutics, Moma Therapeutics, and Aspen Neuroscience raised around $270 million, and 150 million, $147.5 million, respectively. All three of these companies are working on cutting-edge therapies that have the potential to change the lives of patients suffering from these devastating diseases. Apart from this, these company are holding major market share as they have established themselves in the market.

It is clear that biologics research is an area where there is a lot of interest and investment is coming in the global next generation sequencing market. This is good news for patients who are waiting for new treatments and cures. With more funding available, these top companies should be able to make even more progress in finding new ways to treat.

Ultima Genomics is a new biotech company that is working on cutting the cost of sequencing the human genome. Theyve created a powerful technology called named Triple X that adjusts itself by integrating new data and machine learning to bring down the cost. The first use for this companys pitch will be whole-genome sequencing, which can also be applied to other approaches like single-cell and methylation sequencing.

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Key Vendors in Next Generation Sequencing Market

PerkinElmer Inc. (US)

BGI Group (China)

Agilent Technologies Inc. (US)

Eurofins Scientific SE (Luxembourg)

Pacific Biosciences of California Inc. (US)

Oxford Nanopore Technologies (UK)

QIAGEN NV (Netherlands)

F. Hoffmann-La Roche AG (Switzerland)

GENEWIZ Inc. (US)

Psomagen, Inc. (South Korea)

10x Genomics Inc. (US)

Takara Bio (Japan)

Zymo Research (US)

NuGen Technologies (US)

Hamilton Company (US)

Beckman Coulter (US)

Becton, Dickinson, and Company (US)

Lucigen Corporation (US)

Novogene Co., Ltd. (China)

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Next Generation Sequencing Market to Reach $33.73 billion By 2028 Thanks to Increased Attention Early Disease Diagnosis and High Prevalence of Cancer...

Global Whole Genome Amplification Market Forecast 2022-2028, key research on the industry condition of the Whole Genome Amplification is presented together with the best content, definition, expert opinion, SWOT analysis, meaning, and newest development around the world. The Whole Genome Amplification research includes information on industry size, sales, price, revenue, market share, gross margin, growth rate, and cross structure. The study examines the profit made from the sale of this report and technologies across a number of segments, as well as provides a comprehensive table of contents on the Market.

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Segmentation based on Key players

Sigma-Aldrich QIAGEN NV GE Healthcare LGC Group

Segmentation based on Type

Single Cell WGA Kit Complete WGA Kit WGA Reamplification Kit WGA & Chip DNA Kit Others

Segmentation based on Application

Drug Discovery & Development Disease Diagnosis Agriculture & Veterinary Research Forensics Others

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The research examines the Whole Genome Amplification market in-depth, focusing on several factors such as drivers, restraints, opportunities, and threats. Before investing, stakeholders can use this information to make informed judgments. It also enables you to conduct useful competitive research in order to generate marketing ideas for your products. When it comes to customer happiness, its critical to have a clear understanding of whats going on in the market. The general market scenario is accurately described in this research.

Impact Of Covid-19 on Whole Genome Amplification :

COVID-19 is an unprecedented global public health crisis that has impacted practically every business, and its long-term repercussions are expected to have an influence on industry growth during the forecast period. Our continuous study is enhancing our research approach to guarantee that fundamental COVID-19 concerns and potential solutions are included. The research examines COVID-19 in light of changes in consumer behavior and demand, purchasing patterns, supply chain re-routing, market dynamics, and government involvement. The updated study considers the impact of COVID-19 on the market and provides insights, analysis, projections, and forecasts.

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The following geographic segments are covered in the report:

The Whole Genome Amplification report provides information on the market area, which is divided into sub-regions and countries/regions. In addition to the market share in each country and sub-region, this chapter in this report also contains information on profit opportunities. This chapter of the report mentions the market share and growth rate for each region, country, and sub-region during the estimated period.

North America includes the United States, Canada, and Mexico

Europe includes Germany, France, UK, Italy, Spain

South America includes Colombia, Argentina, Nigeria, and Chile

The Asia Pacific includes Japan, China, Korea, India, Saudi Arabia, and Southeast Asia

When analyzing the key market participants, what aspects are taken into account?

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Whole Genome Amplification Market Report: One Research Solution Reveal Everything You Need to Know About Key Players: Sigma-Aldrich, QIAGEN NV, GE...

The discoveries show that ancient DNA can be recovered from Pompeiian human bones, providing new insight into this historic communitys genetic history and lifestyles.

Research that was recently published in Scientific Reports presents the first human genome that has been successfully sequenced from a person who passed away in Pompeii, Italy, after Mount Vesuvius explosion in the year 79 CE. Only little segments of mitochondrial DNA from Pompeiian human and animal remains have been sequenced up to this point.

The DNA of two peoples bones that were discovered in Pompeiis House of the Craftsman was studied and extracted by Gabriele Scorrano and colleagues. The bones length, form, and structure revealed that one pair belonged to a male who was between 35 and 40 years old when he passed away, while the other set belonged to a femalewho was over 50. The authors were able to extract and sequence ancient DNA from both people, but since the sequences from the females bones had gaps in them, they could only sequence the entire genome from the males remains.

The male subjects DNA was compared to 1,030 ancient and 471 current western Eurasian subjects, and it was found that the male subjects DNA was most comparable to that of modern central Italians and other people who resided in Italy during the Roman Imperial era. However, studies of the males Y chromosome and mitochondrial DNA revealed sets of genes that are often prevalent in Sardinian people but not in other people who resided in Italy during the Roman Imperial era. This shows that the Italian Peninsula may have seen high levels of genetic diversity at the time.

Additional analyses of the male individuals skeleton and DNA identified lesions in one of the vertebrae and DNA sequences that are commonly found in Mycobacterium, the group of bacteria that the tuberculosis-causing bacteria Mycobacterium tuberculosis belongs to. This suggests that the individual may have been affected by tuberculosis prior to his death.

The authors speculate that it may have been possible to successfully recover ancient DNA from the male individuals remains as pyroclastic materials released during the eruption may have provided protection from DNA-degrading environmental factors, such as atmospheric oxygen. The findings demonstrate the possibility to retrieve ancient DNA from Pompeiian human remains and provide further insight into the genetic history and lives of this population, they add.

Reference: Bioarchaeological and palaeogenomic portrait of two Pompeians that died during the eruption of Vesuvius in 79 AD by Gabriele Scorrano, Serena Viva, Thomaz Pinotti, Pier Francesco Fabbri, Olga Rickards, and Fabio Macciardi, 26 May 2022, Scientific Reports.DOI: 10.1038/s41598-022-10899-1

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Scientists Have Sequenced the DNA of a 2000-Year-Old Human From Pompeii - SciTechDaily