Linker Histone Functions of HMO1- Implications for DNA repair

The DNA of eukaryotic cells does not exist in free linear strands; it is tightly packaged and wrapped around nuclear proteins in order to be accommodated it inside the nucleus. The basal repeating unit of chromatin, termed the nucleosome, provides the first level of compaction of DNA into the nucleus. Nucleosomes are interconnected by linker DNA and associated linker histones to form 30 nm fibers. The highly diverse linker histones are critical for compaction and stabilization of higher order chromatin structure by binding DNA entering and exiting the nucleosome. The lysine-rich C-terminal domain (CTD) of metazoan H1 is crucial for such stabilization. This study concerns the functions of Saccharomyces cerevisiae Hmo1p, an high mobility group (HMGB) family protein unique in containing a terminal lysine-rich domain and functions in stabilizing genomic DNA. My study suggests that Hmo1p shares with mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of Hmo1p or deletion of the Hmo1p CTD creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin remodeling events associated with DNA double strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of Hmo1p. Further, my data indicates that Hmo1p functions in the DNA damage response by directing lesions towards the error-free pathway. My results suggest that Hmo1p controls DNA end resection and favors the classical non- homologous end joining (NHEJ) over alternate end Joining (A-EJ) that is error-prone process. In all, my study identifies a novel linker histone function of Hmo1p in Saccharomyces cerevisiae with the ability to stabilize genomic DNA, and appears to go beyond conventional linker histone function

The high mobility group protein HMO1 functions as a linker histone in yeast

© 2016 Panday and Grove. Background: Eukaryotic chromatin consists of nucleosome core particles connected by linker DNA of variable length. Histone H1 associates with the linker DNA to stabilize the higher-order chromatin structure and to modulate the ability of regulatory factors to access their nucleosomal targets. In Saccharomyces cerevisiae, the protein with greatest sequence similarity to H1 is Hho1p. However, during vegetative growth, hho1Δ; cells do not show any discernible cell growth defects or the changes in bulk chromatin structure that are characteristic of chromatin from multicellular eukaryotes in which H1 is depleted. In contrast, the yeast high mobility group (HMGB) protein HMO1 has been reported to compact chromatin, as evidenced by increased nuclease sensitivity in hmo1Δ cells. HMO1 has an unusual domain architecture compared to vertebrate HMGB proteins in that the HMG domains are followed by a lysine-rich extension instead of an acidic domain. We address here the hypothesis that HMO1 serves the role of H1 in terms of chromatin compaction and that this function requires the lysine-rich extension. Results: We show here that HMO1 fulfills this function of a linker histone. For histone H1, chromatin compaction requires its basic C-terminal domain, and we find that the same pertains to HMO1, as deletion of its C-terminal lysine-rich extension renders chromatin nuclease sensitive. On rDNA, deletion of both HMO1 and Hho1p is required for significantly increased nuclease sensitivity. Expression of human histone H1 completely reverses the nuclease sensitivity characteristic of chromatin isolated from hmo1Δ cells. While chromatin remodeling events associated with repair of DNA double-strand breaks occur faster in the more dynamic chromatin environment created by the hmo1 deletion, expression of human histone H1 results in chromatin remodeling and double-strand break repair similar to that observed in wild-type cells. Conclusion: Our data suggest that S. cerevisiae HMO1 protects linker DNA from nuclease digestion, a property also characteristic of mammalian linker histone H1. Notably, association with HMO1 creates a less dynamic chromatin environment that depends on its lysine-rich domain. That HMO1 has linker histone function has implications for investigations of chromatin structure and function as well as for evolution of proteins with roles in chromatin compaction

Yeast HMO1: Linker histone reinvented

© 2016 American Society for Microbiology. All Rights Reserved. Eukaryotic genomes are packaged in chromatin. The higher-order organization of nucleosome core particles is controlled by the association of the intervening linker DNA with either the linker histone H1 or high mobility group box (HMGB) proteins. While H1 is thought to stabilize the nucleosome by preventing DNA unwrapping, the DNA bending imposed by HMGB may propagate to the nucleosome to destabilize chromatin. For metazoan H1, chromatin compaction requires its lysine-rich C-terminal domain, a domain that is buried between globular domains in the previously characterized yeast Saccharomyces cerevisiae linker histone Hho1p. Here, we discuss the functions of S. cerevisiae HMO1, an HMGB family protein unique in containing a terminal lysine-rich domain and in stabilizing genomic DNA. On ribosomal DNA (rDNA) and genes encoding ribosomal proteins, HMO1 appears to exert its role primarily by stabilizing nucleosome-free regions or fragile nucleosomes. During replication, HMO1 likewise appears to ensure low nucleosome density at DNA junctions associated with the DNA damage response or the need for topoisomerases to resolve catenanes. Notably, HMO1 shares with the mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of HMO1 creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin-remodeling events associated with DNA double-strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of HMO1. Thus, HMO1 appears to have evolved a unique linker histone-like function involving the ability to stabilize both conventional nucleosome arrays as well as DNA regions characterized by low nucleosome density or the presence of noncanonical nucleosomes

Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

© The Author(s) 2015. DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair

Control of DNA end resection by yeast Hmo1p affects efficiency of DNA end-joining

© 2017 Elsevier B.V. The primary pathways for DNA double strand break (DSB) repair are homologous recombination (HR) and non-homologous end–joining (NHEJ). The choice between HR and NHEJ is influenced by the extent of DNA end resection, as extensive resection is required for HR but repressive to NHEJ. Conversely, association of the DNA end-binding protein Ku, which is integral to classical NHEJ, inhibits resection. In absence of key NHEJ components, a third repair pathway is exposed; this alternative-end joining (A-EJ) is a highly error-prone process that uses micro-homologies at the breakpoints and is initiated by DNA end resection. In Saccharomyces cerevisiae, the high mobility group protein Hmo1p has been implicated in controlling DNA end resection, suggesting its potential role in repair pathway choice. Using a plasmid end-joining assay, we show here that absence of Hmo1p results in reduced repair efficiency and accuracy, indicating that Hmo1p promotes end-joining; this effect is only observed on DNA with protruding ends. Notably, inhibition of DNA end resection in an hmo1Δ strain restores repair efficiency to the levels observed in wild-type cells. In absence of Ku, HMO1 deletion also reduces repair efficiency further, while inhibition of resection restores repair efficiency to the levels observed in kuΔ. We suggest that Hmo1p functions to control DNA end resection, thereby preventing error-prone A-EJ repair and directing repairs towards classical NHEJ. The very low efficiency of DSB repair in kuΔhmo1Δ cells further suggests that excessive DNA resection is inhibitory for A-EJ

DNA damage regulates direct association of TOR kinase with the RNA polymerase II-transcribed HMO1 gene

© 2017 Panday et al. The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient sufficiency and cellular stress. When mTORC1 is inhibited, protein synthesis is reduced in an intricate process that includes a concerted down-regulation of genes encoding rRNA and ribosomal proteins. The Saccharomyces cerevisiae high-mobility group protein Hmo1p has been implicated in coordinating this response to mTORC1 inhibition. We show here that Tor1p binds directly to the HMO1 gene (but not to genes that are not linked to ribosome biogenesis) and that the presence of Tor1p is associated with activation of gene activity. Persistent induction of DNA double-strand breaks or mTORC1 inhibition by rapamycin results in reduced levels of HMO1 mRNA, but only in the presence of Tor1p. This down-regulation is accompanied by eviction of Ifh1p and recruitment of Crf1p, followed by concerted dissociation of Hmo1p and Tor1p. These findings uncover a novel role for TOR kinase in control of gene activity by direct association with an RNA polymerase II-transcribed gene

Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/<i>Ter</i> mammalian replication fork barrier

<div><p>Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/<i>Ter</i> RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/<i>Ter</i>-induced STGC proceeds <i>via</i> a two-ended DSB intermediate. If so, Tus/<i>Ter</i>-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/<i>Ter</i>-induced HR is unaffected by deletion of either of two C-NHEJ genes, <i>Xrcc4</i> or <i>Ku70</i>. These results show that Tus/<i>Ter</i>-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/<i>Ter</i>-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/<i>Ter</i> RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/<i>Ter</i> was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/<i>Ter</i> was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/<i>Ter</i> RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.</p></div

Hypothetical models of Tus/<i>Ter</i>-induced HR.

<p><b>A,</b> Conventional DSB intermediate model. Dual incision of bidirectionally arrested forks generates DNA ends that are processed for HR. Unknown mechanisms prevent Ku access to the DNA ends at the stalled fork. Dark blue: parental strands. Light blue: nascent strands. Half arrows indicate direction of nascent strand synthesis. Orange triangles: Tus/<i>Ter</i> RFB. Green circles: Rad51 monomers. <b>B,</b> Template switch/fork reversal model. Rad51 is loaded onto exposed ssDNA lagging strand daughter strand gaps at the arrested fork. Following replisome disassembly, Rad51 mediates fork remodeling via a template switch mechanism. This process displaces the 3’ ssDNA end of the nascent leading strand, which is rapidly coated with RPA (not shown) followed by Rad51. The DNA end thus generated is incapable of binding Ku, excluding engagement of C-NHEJ. Further processing of the reversed fork may liberate the DNA end by more extensive fork reversal (not shown) and/or <i>via</i> incision of the 4-way reversed fork structure (red arrowhead). Although processing of the two opposing forks is depicted here as sequential, this model is also compatible with synchronous remodeling of both forks. Symbols as in panel A. Pale green circles, Rad51 monomers displaced from lagging strand.</p

Loss of <i>Xrcc4</i> does not perturb HR regulation of Tus/<i>Ter</i>-induced STGC and LTGC.

<p><b>A</b>, Frequencies of Tus/<i>Ter</i>-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector) with selection of transduced cells in 100 μg/mL NTC. Cells were transiently co-transfected with empty or 3xMyc-NLS Tus expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^<b>Δ/Δ</b> <b>clone #11 pHIV-EV</b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0005; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC vs</i>. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0153; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.1481; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0034; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.2292; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0018. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0003; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-<i>mXrcc4</i></b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0002; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001;si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0023; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0240; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0002; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.7398; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0022. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0004; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0049; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0051. <b>B</b>, Frequencies of I-SceI-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control, “EV”) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector, “X4”) with selection of transduced cells in 100 μg/ml NTC. Cells were co-transiently transfected with empty, or 3xMyc-NLS I-SceI expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-EV</b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001 si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.3335; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0006. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0020; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.6260. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone 11 pHIV-<i>mXrcc4</i></b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0002; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0590; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0001. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0011; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0330; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0491; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0017; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0136. <b>C</b>, RT qPCR analysis of <i>BRCA1</i>, BRCA2, <i>CtIP</i> and <i>SLX4</i> mRNA in siRNA-treated <i>Xrcc4</i>^Δ<i>/</i>Δ cells stably transduced with pHIV-EV (“EV”) or pHIV-<i>mXrcc4</i> (“X4”) derived lentivirus. Data normalized to <i>GAPDH</i> and expressed as fold difference from si<i>LUC</i> sample from the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct _target-Ct_Gapdh]-[Ct_si<i>LUC</i>-Ct_{si<i>GAPDH</i>}]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_{<i>TARGET</i>}² + SDEV_<i>GAPDH</i>²]). <b>D</b>, Western blot of RAD51 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”). <b>E</b>, Western blot of Brca1 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”).</p