Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology.

The recent advent of third-generation sequencing technologies brings promise for better characterization of genomic structural variants by virtue of having longer reads. However, long-read applications are still constrained by their high sequencing error rates and low sequencing throughput. Here, we present NanoVar, an optimized structural variant caller utilizing low-depth (8X) whole-genome sequencing data generated by Oxford Nanopore Technologies. NanoVar exhibits higher structural variant calling accuracy when benchmarked against current tools using low-depth simulated datasets. In patient samples, we successfully validate structural variants characterized by NanoVar and uncover normal alternative sequences or alleles which are present in healthy individuals.

CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin–chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter–enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter–enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter–enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter–enhancer loops.

Despite the increasing relevance of structural variants (SV) in the development of many human diseases, progress in novel pathological SV discovery remains impeded, partly due to the challenges of accurate and routine SV characterization in patients. The recent advent of third-generation sequencing (3GS) technologies brings promise for better characterization of genomic aberrations by virtue of having longer reads. However, the applications of 3GS are restricted by their high sequencing error rates and low sequencing throughput. To overcome these limitations, we present NanoVar, an accurate, rapid and low-depth (4X) 3GS SV caller utilizing long-reads generated by Oxford Nanopore Technologies. NanoVar employs split-reads and hard-clipped reads for SV detection and utilizes a neural network classifier for true SV enrichment. In simulated data, NanoVar demonstrated the highest SV detection accuracy (F1 score = 0.91) amongst other long-read SV callers using 12 gigabases (4X) of sequencing data. In patient samples, besides the detection of genomic aberrations, NanoVar also uncovered many normal alternative sequences or alleles which were present in healthy individuals. The low sequencing depth requirements of NanoVar enable the use of Nanopore sequencing for accurate SV characterization at a lower sequencing cost, an approach compatible with clinical studies and largescale SV-association research. ________________________________________________________________________________________

Hematopoietic stem cells (HSC) have the potential to replenish the blood system for the lifetime of the organism. Their two defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Here, using conditional gene knockout models, we demonstrate a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance both in the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 co-localizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell-cycle and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin and Tip60 deletion induced a robust reduction in the acH2A.Z / H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance at least in part through Tip60 dependent H2A.Z acetylation to activate Myc target genes.

Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage-specific transcriptional programs. Structure-specific recognition protein-1 (SSRP1) is a histone chaperone which is important for chromatin-associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage-specific effects by increasing expression of adipocyte-specific genes and decreasing the expression of osteoblast-specific genes. Consistent with a role in controlling lineage specification, transcriptome-wide RNA-sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active b-catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. STEM CELLS 2016;34:1369-1376 SIGNIFICANCE STATEMENTEpigenetic regulation plays an essential role in defining lineage-specific gene expression patterns. We investigated the function of the histone chaperone SSRP1 during cellular differentiation and demonstrate its essential function in specifically directing multipotent mesenchymal stem cell differentiation to the osteoblast lineage via regulation of Wnt signaling. We thereby provide the first evidence of a lineage-specific role of SSRP1 during cellular differentiation and uncover molecular insights into its function, which may serve as a basis for future clinical application of small molecule modulators of its activity for the treatment of aging-related osteoporosis and other diseases associated with altered Wnt signaling.

TIP60 is a lysine acetyltransferase and is known to be a haplo-insufficient tumor suppressor. TIP60 downregulation is an early event in tumorigenesis which has been observed in several cancer types including breast and colorectal cancers. However, the mechanism by which it regulates tumor progression is not well understood. In this study, we identified the role of TIP60 in the silencing of endogenous retroviral elements (ERVs). TIP60-mediated silencing of ERVs is dependent on BRD4. TIP60 and BRD4 positively regulate the expression of enzymes, SUV39H1 and SETDB1 and thereby, the global H3K9 trimethylation (H3K9me3) level. In colorectal cancer, we found that the loss of TIP60 de-represses retrotransposon elements genome-wide, which in turn activate the cellular response to pathogens, mediated by STING, culminating in an induction of Interferon Regulatory Factor 7 (IRF7) and associated inflammatory response. In summary, this study has identified a unique mechanism of ERV regulation in cancer cells mediated by TIP60 and BRD4 through regulation of histone H3 K9 trimethylation, and a new tumor suppressive role of TIP60 in vivo.