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