H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z[superscript AP3]) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z[superscript AP3] interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z[superscript AP3] was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z[superscript AP3] ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z[superscript AP3] ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z[superscript AP3] displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z[superscript AP3] mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.Massachusetts Life Sciences Center (David H. Koch Institute for Integrative Cancer Research at MIT Core Grant P30-CA14051)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511)MIT Faculty Start-up FundMassachusetts Institute of Technology. Computational and Systems Biology Initiative (Merck & Co. Postdoctoral Fellowship

Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types

H2A.Z acidic patch is necessary for ESC differentiation.

<p>(A) Surface rendering of the H2A (left) and H2A.Z (right) nucleosome center. The H2A (orange) and H2A.Z (light brown) structures are shown with H2B (red), H3 (blue), and H4 (green) as indicated by the labels. The divergent residues (teal) are highlighted with arrows. The images were generated in Pymol using the following PDB files: 1AOI for canonical H2A-containing nucleosome structure and 1F66 for H2A.Z nucleosome structure. Below, sequence alignment of C terminal docking domain of H2A and H2A.Z. The acidic patch region is highlighted in a grey box. The bold, italicized and underlined residues indicate the divergent H2A.Z residues replaced in our study to the corresponding H2A residues. (B) Schematic diagram depicting the system used in this study to investigate the function of the H2A.Z acidic patch. (C) qRT-PCR representing the relative levels of endogenous H2A.Z transcript in H2A.Z^WT and H2A.Z^AP3 dox-inducible transgenic ESCs in the presence (+) and absence (−) of H2A.Z 3′UTR-specific shRNA. Transcript levels were normalized relative to Tubb5. Error bars represents standard deviation calculated from three independent biological replicates. (D) (Top) Western blot using H2A.Z antibodies on whole cell lysates isolated from dox-induced and uninduced (−/+) H2A.Z^WT and H2A.Z^AP3 transgenic ESC lines in the presence (+) and absence (−) of the H2A.Z 3′UTR-specific shRNA. Titrations of the dox-induced samples (25 µg and 50 µg of whole cell lysates) were performed to demonstrate comparable expression of H2A.Z-YFP transgene in H2A.Z^WT and H2A.Z^AP3 ESCs. H3 levels were used as a load control (lower exposure). Densitometric measurements were used to determine the ratio of transgene signal to H3 for the indicated samples (bottom) using ImageJ. Error bars represent standard deviations from a triplicate set of experiments. ESC colony morphology and OCT4 staining for H2A.Z^WT, H2A.Z^KD, (E) and H2A.Z^AP3 (G) expressing ESC. Embryoid bodies (EBs) were generated from H2A.Z^WT, H2A.Z^KD (F), and H2A.Z^AP3 (H) expressing ESCs. The left panels show bright field images of EBs cultured for 10 days in the absence of LIF. The right panels show hematoxylin and eosin stained sections of day 10 EBs for indicated cell lines. (I) qRT-PCR analyses showing mRNA levels of indicated pluripotency (<i>Pou5f1/Oct4</i> and <i>Nanog</i>) and differentiation markers (<i>Nestin</i>, <i>Cdx2</i>, <i>Isl1</i>) in Day 0 and Day 10 EBs generated from unmodified ESCs, H2A.Z^WT, H2A.Z^KD, and H2A.Z^AP3 ESCs. Student's t-test was performed to generate indicated p values. ***p<0.005, **p<0.01, *p<0.05. Error bars represent standard deviations from a triplicate set of experiments.</p

H2A.Z acidic patch couples chromatin dynamics to gene expression regulation during ESC differentiation.

<p>Schematic depicting H2A.Z dynamics in H2A.Z^WT (A) and H2A.Z^AP3 (B) ESCs and its consequence on gene expression of developmental genes and ultimately lineage commitment. The black arrow represents similar on rate for H2A.Z -containing dimers in H2A.Z^WT and H2A.Z^AP3 ESCs while a bold black arrow represents a greater off rate for H2A.Z^AP3-H2B dimers whereas the hashed black arrow represents weaker off rate for H2A.Z-H2B dimers in H2A.Z^WT. The model demonstrates how the divergent acidic patch domain functions to couple H2A.Z dynamics with the regulation of gene expression programs during ESC differentiation.</p

Disruption of H2A.Z acidic patch increases chromatin dynamics in a transcription-dependent manner.

<p>(A) qRT-PCR results showing a decrease in the transcript levels of active genes- <i>Oct4</i>, <i>Tubb5</i>, and <i>Actin</i>, upon treatment of 1 µM flavopiridol and restoration of transcript levels upon removal of the agent at the various time points in H2A.Z^WT ESCs. (B–D) Graphs representing the estimated mobile fractions in H2A (B), H2A.Z^WT (C), and H2A.Z^AP3 (D) expressing ESCs upon treatment and removal of flavopiridol. ESCs treated with DMSO were used as a control. P values were generated by standard unpaired Student's t-test. *p<0.03, **p<0.015 and NS indicates p values >0.6. Quantitative PCR on ChIP DNA generated by using GFP antibodies in H2A.Z^AP3 (E) and H2A.Z^WT (F) ESCs treated with DMSO, 1 µM flavopiridol (Flavo) and 2 hrs after flavopiridol removal (Wash). Student's t-test was performed to test for statistical significance. * p<0.03, ** p<0.01, and NS represents not significant (>0.05). Error bars represent standard deviations from a triplicate set of experiments.</p