We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.
Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis1,2. Vessel normalization (VN) may resolve this paradox3. VN involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia3. While these processes alter tumour progression, their regulation is poorly understood. Here we show that Type 1 T helper (Th1) cells play a crucial role in VN. Bioinformatic analyses revealed that gene expression features related to VN correlate with immunostimulatory pathways, especially T lymphocyte (TL) infiltration/activities. To delineate the causal relationship, we employed various mouse models with VN or TL deficiencies. While VN disruption reduced TL infiltration as expected4, reciprocal depletion or inactivation of CD4+-TLs decreased VN, indicating a mutually-regulatory loop. Additionally, CD4+-TL activation by immune checkpoint blockade (ICB) increased VN. IFNγ+ Th1 cells are the major population associated with VN. Patient-derived xenograft (PDX) tumours growing in immunodeficient animal hosts exhibited enhanced hypoxia compared to the original tumours in immunocompetent human hosts, which was reduced by adoptive Th1 transfer. Our findings elucidate an unexpected role of Th1 in vasculature and immune reprogramming. Th1 cells may be a marker and a determinant of both ICB and anti-angiogenesis efficacies.
Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.
Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-alpha (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 approximately 5-6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.
Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2 ؊/؊ mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance, and hepatic steatosis. In vitro differentiation of both Bscl2
Spinal bulbar muscular atrophy is a neurodegenerative disorder caused by a polyglutamine expansion in the androgen receptor (AR). We show in transiently transfected HeLa cells that an AR containing 48 glutamines (ARQ48) accumulates in a hormone-dependent manner in both cytoplasmic and nuclear aggregates. Electron microscopy reveals both types of aggregates to have a similar ultrastructure. ARQ48 aggregates sequester mitochondria and steroid receptor coactivator 1 and stain positively for NEDD8, Hsp70, Hsp90 and HDJ-2/HSDJ. Co-expression of HDJ-2/HSDJ significantly represses aggregate formation. ARQ48 aggregates also label with antibodies recognizing the PA700 proteasome caps but not 20S core particles. These results suggest that ARQ48 accumulates due to protein misfolding and a breakdown in proteolytic processing. Furthermore, the homeostatic disturbances associated with aggregate formation may affect normal cell function.
Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation. C ancers of the female reproductive system are serious human health problems, and estrogen plays a critical role in the initiation and progression of these diseases (1). Despite decades of research into mechanisms of 17β-estradiol (E2)-responsive gene transcription, our understanding of this process is far from complete (2). It is generally believed that, upon E2 binding, the nuclear hormone receptor estrogen receptor α (hereafter called ER) undergoes major structural reorganization, associates with estrogen-response elements (ERE) within target gene promoters, and recruits a range of coactivators including histone modification enzymes (3-6). After deposition, the resulting histone modifications can then modulate target gene activity by affecting local chromatin structure and regulating the accessibility of chromatin to transcription factors (2, 5, 7-9).Peptidylarginine deiminase (PAD) enzymes convert arginine and methylarginine residues to citrulline via a hydrolytic process termed citrullination or deimination (10, 11). We and others have shown that one such PAD, PAD4, appears to play a repressive role in regulating the expression of the canonical ER target gene, TFF1, via citrullination of histone H4 methylarginine 3, thus suggesting that PADs potentially function as ER cofactors (12, 13). Given that these previous studies were limited to a single ER target promoter, we chose to take a more comprehesive approach to test whether PAD-mediated histone tail citrullination may be more fundamental to ER target gene regulation than previously realized. In this study, we show that citrullination of histone H3R26 at ER targets is closely associated with gene transcription and that citrullination at this residue is catalyzed by PAD2, as opposed to PAD4. Additionally, we show that PAD2 interacts with ER and that PAD2-mediated citrullination of H3R26 likely facilitates transcriptional activation by creating an open, permissive, chromatin architecture around the EREs of E2-i...
A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named ''mitosin,'' was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G 2 , and M phases of the cell cycle but is absent in G 1 . Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G 2 /M. These results suggest that mitosin may play an important role in mitotic-phase progression.
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