Increasing evidence suggests that linker histone H1 can influence distinct cellular processes by acting as a gene-specific regulator. However, the mechanistic basis underlying such H1 specificity and whether H1 acts in concert with other chromatin-altering activities remain unclear. Here, we show that one of the H1 subtypes, H1.2, stably interacts with Cul4A E3 ubiquitin ligase and PAF1 elongation complexes, and that such interaction potentiates target gene transcription via induction of H4K31ubiquitylation, H3K4me3 and H3K79me2. H1.2, Cul4A and PAF1 are functionally cooperative, as their individual knockdown results in the loss of the corresponding histone marks and the deficiency of target gene transcription. H1.2 interacts with the serine 2 phosphorylated form of RNAPII and we argue that it recruits the Cul4A and PAF1 complexes to target genes by bridging the interaction between the and the Cul4A and PAF1 complexes. These data define an expanded role for H1 in regulating gene transcription and illustrate its dependence on the elongation competence of RNAPII.
Histone; Chromatin; Transcription; Methylation; Ubiquitylation; H1; PAF1; Cul4A
The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.
Past studies have documented a cross-talk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little, if any, direct evidence exists explaining the mechanism underlying H2Bub-dependent H3K4 methylation on chromatin templates. Here, we took advantage of an in vitro histone methyltransferase assay employing a reconstituted yeast Set1 complex (ySet1C) and a recombinant chromatin template containing fully ubiquitylated H2B to gain valuable insights. Combined with genetic analyses, we demonstrate that the n-SET domain within Set1, but not Swd2, is essential for H2Bub-dependent H3K4 methylation. Spp1, a homolog of human CFP1, is conditionally involved in this cross-talk. Our findings extend to the human Set1 complex, underscoring the conserved nature of this disease-relevant, cross-talk pathway. As not all members of the H3K4 methyltransferase family contain n-SET domains, our studies call attention to the n-SET domain as being a predictor of H2B ubiquitylation ‘sensing’ in bringing about downstream H3K4 methylation.
Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans1. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the ‘histone code’ hypothesis, in which histone modifications are integral to choreographing the expression of the genome1,2. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans3, and lysine 123 in yeast4, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5–8), by K79-specific methyltransferase Dot1 (KMT4)9–11. However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.
Viral infection is commonly associated with virus-driven hijacking of host proteins. Here we describe a novel mechanism by which influenza virus affects host cells through the interaction of influenza non-structural protein 1 (NS1) with the infected cell epigenome. We show that the NS1 protein of influenza A H3N2 subtype possesses a histone-like sequence (histone mimic) that is used by the virus to target the human PAF1 transcription elongation complex (hPAF1C). We demonstrate that binding of NS1 to hPAF1C depends on the NS1 histone mimic and results in suppression of hPAF1C-mediated transcriptional elongation. Furthermore, human PAF1 has a crucial role in the antiviral response. Loss of hPAF1C binding by NS1 attenuates influenza infection, whereas hPAF1C deficiency reduces antiviral gene expression and renders cells more susceptible to viruses. We propose that the histone mimic in NS1 enables the influenza virus to affect inducible gene expression selectively, thus contributing to suppression of the antiviral response.
Diverse histone modifications play important roles in transcriptional regulation throughout eukaryotes, and recent studies have implicated histone H2B ubiquitylation in active transcription. The necessity of at least three enzymes (E1, E2 and E3), as well as ongoing transcription events, for efficient H2B ubiquitylation complicates mechanistic studies of H2B ubiquitylation relative to other histone modifications. Here we describe experimental protocols for preparation of human H2B ubiquitylation factors, ubiquitylation substrates and transcription factors, as well as the use of these factors to establish H2B ubiquitylation mechanisms during transcription. The methods include reliable protein interaction and E3 ubiquitylation assays that can be widely applied to confirm cognate E2–E3 pairs in other protein ubiquitylation systems, optimized in vitro ubiquitylation assays for various histone substrates, and a transcription-coupled H2B ubiquitylation assay in a highly purified transcription system. These comprehensive analyses have revealed (i) that RAD6 serves as the cognate E2 for the BRE1 complex in human cells, as previously established in yeast, (ii) that RAD6, through direct interaction with the BRE1 complex, ubiquitylates chromatinized H2B at lysine 120 and (iii) that PAF1 complex-mediated transcription is required for efficient H2B ubiquitylation. This experimental system permits detailed mechanistic analyses of H2B ubiquitylation during transcription by providing information concerning both precise enzyme functions and physical interactions between the transcription and histone modification machineries.
Histone H2B ubiquitylation; RAD6; BRE1 complex; PAF1 complex; Chromatin; Transcription
hBRE1/RNF20 is the major E3 ubiquitin ligase for histone H2B. RNF20 depletion causes a global reduction of monoubiquitylated H2B (H2Bub) levels and augments the expression of growth promoting, pro-oncogenic genes. Those genes reside preferentially in compact chromatin, and are inefficiently transcribed under basal conditions. We now report that RNF20, presumably via H2Bub, represses selectively those genes by interfering with chromatin recruitment of TFIIS, a factor capable of relieving stalled RNA polymerase II. RNF20 inhibits the interaction between TFIIS and the PAF1 complex and hinders transcriptional elongation. TFIIS ablation selectively abolishes the upregulation of those genes upon RNF20 depletion, and attenuates the cellular response to EGF. Consistent with its positive role in transcription of pro-oncogenic genes, TFIIS expression is elevated in various human tumors. Our findings provide a molecular mechanism for selective gene repression by RNF20, and position TFIIS as a key target of RNF20's tumor suppressor activity.
A 1-year-old female mongrel dog was evaluated for anorexia and vomiting of 4 days duration. Abdominal ultrasonographic findings revealed small kidneys with multiple anechoic cysts. The dog was euthanized due to poor prognosis. A full necropsy was performed, and the histopathologic findings were consistent with multicystic dysplastic kidney disease.
MLL1 fusion proteins activate HoxA9 gene expression and cause aggressive leukemias that respond poorly to treatment, but how they recognize and stably bind to HoxA9 is not clearly understood. In a systematic analysis of MLL1 domain recruitment activity, we identified an essential MLL1 recruitment domain that includes the CXXC domain and PHD fingers and is controlled by direct interactions with the PAF elongation complex and H3K4Me2/3. MLL1 fusion proteins lack the PHD fingers and require pre-binding of a wild type MLL1 complex and CXXC domain recognition of DNA for stable HoxA9 association. Together, these results suggest that specific recruitment of MLL1 requires multiple interactions and is a precondition for stable recruitment of MLL1 fusion proteins to HoxA9 in leukemogenesis. Since wild type MLL1 and oncogenic MLL1 fusion proteins have overlapping yet distinct recruitment mechanisms, this creates a “window of opportunity” that could be exploited for the development of targeted therapies.
Genetic and cell-based studies have implicated the PAF1 complex (PAF1C) in transcription-associated events, but there has been no evidence showing a direct role in facilitating transcription of a natural chromatin template. Here, we demonstrate an intrinsic ability of human PAF1C (hPAF1C) to facilitate activator (p53)- and histone acetyltransferase (p300)-dependent transcription elongation from a recombinant chromatin template in a biochemically defined RNA polymerase II transcription system. This represents a PAF1C function distinct from its established role in histone ubiquitylation and methylation. Importantly, we further demonstrate a strong synergy between hPAF1C and elongation factor SII/TFIIS and an underlying mechanism involving direct hPAF1C-SII interactions and cooperative binding to RNA polymerase II. Apart from a distinct PAF1C function, the present observations provide a molecular mechanism for the cooperative function of distinct transcription elongation factors in chromatin transcription.
This study was conducted to examine recent trends in ovarian cancer incidence and mortality and secular trends in demographic factors in Korea.
With the data from Korea Central Cancer Registry, International Agency for Research on Cancer, Korean Death Registry, and World Health Organization's Statistical Information System, we calculated age-standardized incidence and mortality rates for ovarian cancer. Also we estimated future incidence of ovarian and cervical cancer using linear regression model. To assess the demographic trend, data from national surveys in Korea or results from published papers were searched.
Ovarian cancer incidence rate was similar to that in women worldwide but lower than those in Western countries, and the trend has been increased steadily. Ovarian cancer-related mortality rates have been increasing in Korea, even though those in western and some Asian countries, such as China, have been decreasing. Age-specific incidence rate and mortality rate showed steep increases with advancing age. The incidence rate of ovarian cancer was estimated to surpass that of uterine cervix cancer in 2015. Korea showed rapid changes in nutritional, reproductive, and anthropometric factors.
These recent trends in ovarian cancer incidence and mortality may be partly attributed to gradual westernizing of life styles and to changes in socio-demographic behavior factors. In particular, the increasing trend in ovarian cancer mortality in Korea may be attributed to a real rise in mortality as well as, in part, a decline in misclassification bias related to an increase in the proportion of deaths confirmed by physician diagnosis.
Ovarian neoplasms; Epidemiology; Incidence; Mortality
In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged DNA. In this regard, a fundamental question concerns the nature and mechanism of action of the accessory factors that are necessary and sufficient for, or enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we describe a highly purified system that allows for efficient activator-dependent transcription by RNA polymerase II from the promoter through several contiguous nucleosomes on defined chromatin templates. The system contains natural or recombinant histones, chromatin assembly factors, the histone-acetyltranferase p300, all components of the general transcription machinery, general coactivators and the elongation factor SII (TFIIS). As examples of the applicability of this system for mechanistic analyses of these and other factors, representative experiments show (i) that activated transcription from chromatin templates is concomitantly dependent on the activator, p300-mediated histone acetylation and elongation factor SII/TFIIS. (ii) that SII/TFIIS acts in a highly synergistic manner with p300 (and histone acetylation) at a step subsequent to preinitiation complex (PIC) formation and (iii) that SII/TFIIS works directly at the elongation step of chromatin transcription. Here we describe purification methods for the different factors employed and the specific transcriptional assays that led to the above-mentioned conclusions. This purified system will be very useful as an assay system for the discovery of new factors or the mechanistic analysis of known or candidate factors involved in transcription initiation or elongation on chromatin templates, including factors that effect specific histone modifications or nucleosomal remodeling.
Chromatin; Transcription; RNA polymerase II; Elongation
H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (i) that hRAD6 serves as the cognate E2 conjugating enzyme, (ii) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120, (iii) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery, (iv) that H2B ubiquitylation per se does not affect the level of hPAF-, SII- and p300-dependent transcription and likely functions downstream and (v) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and tri-methylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.
Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles.
Placenta increta is an uncommon and life-threatening complication of pregnancy characterized by complete or partial absence of the decidua basalis. Placenta increta usually presents with vaginal bleeding during difficult placental removal in the third-trimester. Although placenta increta may complicate first and early second-trimester pregnancy loss, the diagnosis can be very difficult during early pregnancy and thus the lesion is difficult to identify. We encountered with a woman who was diagnosed with placenta increta after receiving emergency hysterectomy due to intraperitoneal bleeding 2 months after an uncomplicated dilatation and curettage in the first trimester. Therefore, we report this case with a brief review of the literature.
Placenta Increta; First Trimester; Intraperitoneal Bleeding
Transcription in eukaryotes is governed in part by histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-containing complexes that are recruited via activators and repressors, respectively. Here, we show that the Sin3/HDAC and N-CoR/SMRT corepressor complexes repress transcription from histone H3- and/or H4-acetylated nucleosomal templates in vitro. Repression of histone H3-acetylated templates was completely dependent on the histone deacetylase activity of the corepressor complexes, whereas this activity was not required to repress H4-acetylated templates. Following deacetylation, both complexes become stably anchored in a repressor-independent manner to nucleosomal templates containing hypoacetylated histone H3, but not H4, resulting in dominance of repression over activation. The observed stable anchoring of corepressor complexes casts doubt on the view of a dynamic balance between readily exchangeable HAT and HDAC activities regulating transcription and implies that pathways need to be in place to actively remove HDAC complexes from hypoacetylated promoters to switch on silent genes.
In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1–Cul1–F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCFhFBH1 complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCFhFBH1 complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCFhFBH1 complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCFhFBH1 complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCFhFBH1 complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCFhFBH1 complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.