A disordered region controls cBAF activity via condensation and partner recruitment

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct sequence grammars underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved

Oligonucleotide Sequence Motifs as Nucleosome Positioning Signals

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3–4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes

Are nucleosome positions in vivo primarily determined by histone–DNA sequence preferences?

ABSTRACT: Large-scale and genome-wide studies have concluded that ∼80% of the yeast (Saccharomyces cerevisiae) genome is occupied by positioned nucleosomes. In vivo this nucleosome organization can result from a variety of mechanisms, including the intrinsic DNA sequence preferences for wrapping the DNA around the histone core. Recently, a genome-wide study was reported using massively parallel sequencing to directly compare in vivo and in vitro nucleosome positions. It was concluded that intrinsic DNA sequence preferences indeed have a dominant role in determining the in vivo nucleosome organization of the genome, consistent with a genomic code for nucleosome positioning. Some other studies disagree with this view. Using the large amount of data now available from several sources, we have attempted to clarify a fundamental question concerning the packaging of genomic DNA: to what extent are nucleosome positions in vivo determined by histone-DNA sequence preferences? We have analyzed data obtained from different laboratories in the same way, and have directly compared these data. We also identify possible problems with some of the experimental designs used and with the data analysis. Our findings suggest that DNA sequence preferences have only small effects on the positioning of individual nucleosomes throughout the genome in vivo

HIV chromatin is a preferred target for drugs that bind in the DNA minor groove.

The HIV genome is rich in A but not G or U and deficient in C. This nucleotide bias controls HIV phenotype by determining the highly unusual composition of all major HIV proteins. The bias is also responsible for the high frequency of narrow DNA minor groove sites in the double-stranded HIV genome as compared to cellular protein coding sequences and the bulk of the human genome. Since drugs that bind in the DNA minor groove disrupt nucleosomes on sequences that contain closely spaced oligo-A tracts which are prevalent in HIV DNA because of its bias, it was of interest to determine if these drugs exert this selective inhibitory effect on HIV chromatin. To test this possibility, nucleosomes were reconstituted onto five double-stranded DNA fragments from the HIV-1 pol gene in the presence and in the absence of several minor groove binding drugs (MGBDs). The results demonstrated that the MGBDs inhibited the assembly of nucleosomes onto all of the HIV-1 segments in a manner that was proportional to the A-bias, but had no detectable effect on the formation of nucleosomes on control cloned fragments or genomic DNA from chicken and human. Nucleosomes preassembled onto HIV DNA were also preferentially destabilized by the drugs as evidenced by enhanced nuclease accessibility in physiological ionic strength and by the preferential loss of the histone octamer in hyper-physiological salt solutions. The drugs also selectively disrupted HIV-containing nucleosomes in yeast as revealed by enhanced nuclease accessibility of the in vivo assembled HIV chromatin and reductions in superhelical densities of plasmid chromatin containing HIV sequences. A comparison of these results to the density of A-tracts in the HIV genome indicates that a large fraction of the nucleosomes that make up HIV chromatin should be preferred in vitro targets for the MGBDs. These results show that the MGBDs preferentially disrupt HIV-1 chromatin in vitro and in vivo and raise the possibility that non-toxic derivatives of certain MGBDs might serve as a novel class of anti-HIV agents

The Baf Subunit Dpf2 Regulates Resolution of Inflammation By Controlling Macrophage Differentiation Transcription Factor Networks

To mount an effective immune response against infectious pathogens or tissue injury, hematopoietic stem cells (HSCs) increase their proliferation and production of myeloid cells, including macrophages, which destroy the pathogens and repair the damaged tissue. Proper resolution of inflammation is essential to restore hematopoietic homeostasis, as unrestrained inflammation can result in life-threatening pathologies such as sepsis, autoimmune disorders and cancer. The molecular mechanisms that control the resolution of inflammation, and how these contribute to disease phenotypes, are poorly understood. BAF (SWI/SNF) complexes are ATPase dependent chromatin-remodeling complexes that play fundamental roles in transcription. BAF complexes use the energy of ATP to modulate the accessibility of transcription factors to DNA and thus, orchestrate the gene expression programs that control proliferation and cellular identity. Genes encoding for BAF subunits are frequently mutated in cancer and developmental disorders. In hematopoietic malignancies, loss-of-function mutations and low expression of specific BAF subunits have been reported in patients with anemia and bone marrow failure. Work from our lab previously demonstrated that the hematopoietic-specific BAF complex subunit Dpf2 cooperates with the transcription factor Runx1 to regulate myeloid differentiation. Based on these studies, we generated a hematopoietic-specific Dpf2 knock-out mouse model and found that mice lacking Dpf2 develop pancytopenia, anemia and an uncontrolled inflammatory response that results in early death. Dpf2-/- peripheral blood samples showed dysplastic features including increased number of polychromatophilic blood cells and Howell-Jolly bodies in erythrocytes. Histopathological analyses revealed the presence of fibrosis and prominent infiltration of histiocytes in multiple organs, including lungs, liver and spleen. Detailed chemical profiling of plasma showed increased levels of multiple pro-inflammatory cytokines, indicative of systemic inflammation. Flow cytometry analyses and Mass cytometry profiling further revealed an expansion of myeloid lineages, specifically monocytes and macrophages, concomitant with severe defects in lymphoid and erythroid differentiation. We also found that Dpf2-/-HSCs had increased serial re-plating capacity and a marked myeloid differentiation bias. To identify the transcription factor networks underlying these phenotypes, we performed RNAseq and ATACseq on control and Dpf2-/- HSCs. Gene Set Enrichment Analyses indicated that Dpf2-/- HSCs have extensive gene expression alterations in immune signaling and interferon response pathways, as well as leukocyte and erythroid differentiation. We also found that Dpf2 loss results in pronounced changes in expression and genomic accessibility of specific transcription factors that control macrophage differentiation and proliferation. Together, our mechanistic studies support a model whereby the absence of Dpf2 results in misregulation of the transcription factor networks that establish macrophage cell identity, leading to a marked increase in macrophage infiltrations and shortened survival of the mice. Treatment of the Dpf2-/-mice with clodronate-containing liposomes, which deplete macrophages from bone marrow and spleen, prolonged survival of the mice. Our work uncovers a novel role of Dpf2 in restraining inflammatory responses by controlling macrophage proliferation and function. Moreover, we propose that, in addition to their tumor suppressive roles in cancer, BAF complexes may have a central role in the prevention of immunopathologies. Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Scientific founder, fiduciary board of directors member, scientific advisory board member, shareholder, and consultant for Foghorn Therapeutics (Cambridge, MA). . Vega:NCI: Research Funding

PAF1 regulation of promoter-proximal pause release via enhancer activation

Gene expression in metazoans is regulated by RNA polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we showed that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we found that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of PAF1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner