Biology subjects

Billings, T.

Publications and source records attributed to Billings, T..

4 recordsLinked to original sources

bioRxiv2021

Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudo-modularity

PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, glu360pro (Prdm9EP) and glu360lys (Prdm9EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9EP is slightly less hypomorphic than Prdm9EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9EP, but not Prdm9EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9EP and Prdm9EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain versus the full-length protein show that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

genetics↗

bioRxiv2019

Why Do Long Zinc Finger Proteins have Short Motifs? A case study of ZFY and CTCF reveals non-independent recognition of tandem zinc finger proteins.

The human genome contains around 800 C2H2 Zinc Finger Proteins (ZFPs), and many of them are composed of long tandem arrays of zinc fingers. Current motif prediction models assume longer finger arrays correspond to longer DNA-binding motifs and higher specificity. However, recent experimental efforts to identify ZFP binding sites in vivo contradict this assumption, with many having short motifs. Here, we systematically test how multiple zinc fingers contribute to binding for three model ZFPs: Zinc Finger Y (ZFY), CTCF, and ZNF343. Using ZFY, which contains 13 fingers, we quantitatively characterize its binding specificity with several methods, including Affinity-seq, HT-SELEX, Spec-seq and fluorescence anisotropy, and find evidence for dependent recognition where downstream fingers can recognize some extended motifs only in the presence of an intact core site. For the genomic insulator CTCF, additional high-throughput affinity measurements reveal that its upstream specificity profile depends on the strength of the core, violating presumed additivity and positionindependence. Moreover, the effect of different epigenetic modifications within the core site depends on the strength of flanking upstream site, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates CTCFs methylation sensitivity. Lastly, we used ZNF343 as example to show that a simple iterative motif analysis strategy based on a small set of prefixed cores can reveal the dependent relationship between cores and upstream motifs. These results establish that the current underestimation of ZFPs motif lengths is due to our lack of understanding of intrinsic properties of tandem zinc finger recognition, including irregular motif structure, variable spacing, and dependent recognition between sub-motifs. These results also motivate a need for better recognition models beyond additive, position-weight matrix to predict ZFP specificities, occupancies, and the molecular mechanisms of disease mutations.

genetics↗

bioRxiv2018

EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 lysine-4 and lysine-36 methyltransferase PRDM9 to ensure successful double-strand break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse models to deplete EWSR1 before the onset of meiosis, and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis. Author SummaryIn most mammals, including humans and mice, genetic recombination initiates when the meiosis-specific protein PRDM9 binds specific DNA sequences, known as hotspots, at the beginning of the extended prophase I of meiosis, and activates them by trimethylating histone 3 at lysine-4 and lysine-36 on nearby nucleosomes. Although this activation of hotspots is believed to occur on the chromatin loops, the subsequent double-strand break formation and repair occur on a proteinaceous structure known as the chromosome axis. We now show that Ewing sarcoma RNA binding protein 1 (EWSR1) is a key player in early recombination events, binding to PRDM9, promoting PRDM9-dependent histone methylation, and facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through the meiosis-specific cohesion REC8. As a result of these activities, EWSR1 assures sufficient numbers of properly positioned crossovers in each meiosis.

developmental biology↗

bioRxiv2018

CXXC1 is redundant for normal DNA double-strand break formation and meiotic recombination in mouse

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break initiation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here we investigated whether CXXC1 has a similar function in mouse meiosis. We found that CXXC1 is co-expressed and interacts with PRDM9 in mouse spermatocytes. To investigate the meiotic function of CXXC1, we created a Cxxc1 conditional knockout mouse to deplete CXXC1 before the onset of meiosis. Surprisingly, knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation activity, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.\n\nAuthor SummaryMeiotic recombination increases genetic diversity by ensuring novel combination of alleles passing onto the next generation correctly. In most mammals, the meiotic recombination sites are determined by histone methyltransferase PRDM9. These sites subsequently become associated with the chromosome axis with the participation of additional proteins and undergo double strand breaks, which are repaired by homologous recombination. In Saccharomyces cerevisiae, Spp1 (ortholog of CXXC1) binds to methylated H3K4 and connects these sites with chromosome axis promoting DSB formation. However, our data suggest that even though CXXC1 interacts with PRDM9 in male germ cells, it does not play a crucial role in mouse meiotic recombination. These results indicate that, unlike in S. cerevisiae, a recombination initiation pathway that includes CXXC1 could only serve as a non-essential pathway in mouse meiotic recombination.

cell biology↗