Naturally arising CD25+CD4+ regulatory T cells (T(R) cells) are engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. T(R) cells specifically express the transcription factor Foxp3, a key regulator of T(R)-cell development and function. Ectopic expression of Foxp3 in conventional T cells is indeed sufficient to confer suppressive activity, repress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and upregulate T(R)-cell-associated molecules such as CD25, cytotoxic T-lymphocyte-associated antigen-4, and glucocorticoid-induced TNF-receptor-family-related protein. However, the method by which Foxp3 controls these molecular events has yet to be explained. Here we show that the transcription factor AML1 (acute myeloid leukaemia 1)/Runx1 (Runt-related transcription factor 1), which is crucially required for normal haematopoiesis including thymic T-cell development, activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural T(R) cells, Foxp3 interacts physically with AML1. Several lines of evidence support a model in which the interaction suppresses IL-2 and IFN-gamma production, upregulates T(R)-cell-associated molecules, and exerts suppressive activity. This transcriptional control of T(R)-cell function by an interaction between Foxp3 and AML1 can be exploited to control physiological and pathological T-cell-mediated immune responses.

Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfbeta, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfbeta heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10 4 and 10 5 clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.retrovirus | latency | epigenetics | HTLV-1 | CTCF

MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited familial cancer syndrome characterized by the combined occurrence of tumors of the parathyroid gland, the pancreas, and the pituitary gland (2, 38). The responsible gene, MEN1, has been localized to chromosome 11q13 (21) and identified by positional cloning (7). The loss of heterozygosity of the MEN1 locus in tumors suggests that MEN1 is a tumor suppressor gene. MEN1 contains 10 exons and encodes a 610-amino-acid protein, called menin (7), which shows no similarity to any known protein. Previous studies have revealed that menin is found predominantly in the nucleus, contains two independent nuclear localization signals in the C terminus (amino acids 479 to 497 and 588 to 608) (14), and binds to transcription factors, including JunD (1), Smad3 (18) and NF-B (15), all of which suggest a role related to transcriptional regulation. Menin has also been shown to interact with the putative tumor metastasis suppressor/nucleoside diphosphate kinase nm23 (27), the homeobox protein Pem (23), GFAP and vimentin (24), and the 32-kDa subunit (RPA2) of replication protein A (33). However, the exact molecular mechanism for tumor suppression by menin is largely unknown.More than 400 independent germ line mutations distributed throughout the MEN1 coding region have been identified for familial and sporadic cases of MEN1 (13, 36). The majority of these are nonsense mutations or frame shifts, but missense mutations and short in-frame deletions-insertions have also been identified in about 30% of cases. Germ line MEN1 mutations are also found in a small subset of familial isolated hyperparathyroidism (FIHP), a syndrome consisting of genetically heterogeneous diseases that are characterized ...

Strong (10-T) SMFs have no effect on cell growth, cell cycle distribution, or micronucleus frequency, but they may cause an increase in the micronucleus formation induced by 4-Gy x rays.

Molecular diversity through alternative splicing is important for cellular function and development. However, little is known about the factors that regulate alternative splicing. Here we demonstrate that one isoform of coactivator-associated arginine methyltransferase 1 (named CARM1-v3) associates with the U1 small nuclear RNP-specific protein U1C and affects 5 splice site selection of the pre-mRNA splicing. CARM1-v3 was generated by the retention of introns 15 and 16 of the primary transcript of CARM1. Its deduced protein lacks the C-terminal domain of the major isoform of CARM1 and instead has v3-specific sequences at the C terminus. CARM1-v3, but not the other isoforms, strongly stimulates a shift to the distal 5 splice site of the pre-mRNA when the adenoviral E1A minigene is used as a reporter and enhances the exon skips in the CD44 reporter. A CARM1-v3 mutant lacking the v3-specific sequences completely lost the ability to regulate the alternative splicing patterns. In addition, CARM1-v3 shows tissuespecific expression patterns distinct from those of the other isoforms. These results suggest that the transcriptional coactivator can affect the splice site decision in an isoform-specific manner.It has been estimated that about 60% of human genes undergo alternative splicing (1). Commonly, alternative splicing determines the inclusion of a portion of coding sequence in the mRNA, giving rise to protein isoforms that differ in their peptide sequence and hence chemical and biological activity. The mechanism of alternative splicing permits diversity of translatable mRNAs, thereby increasing the proteome diversity encoded by a limited number of genes. Genetic switches based on alternative splicing are known to be important in many cellular and developmental processes, including sex determination, apoptosis, axon guidance, and tissue-specific differentiation (2-4).Although a large variety of splicing decisions can be explained by the antagonistic effects of general splicing factors, such as serine/arginine-rich proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), 1 it is most likely that tissue-specific or developmentally regulated splicing factors have an important role in the regulation of alternative splicing. In Drosophila melanogaster, sex-lethal functions as a regulator of alternative splicing in sex determination (2), and the embryonic lethal abnormal visual system is a gene-specific regulator of alternative pre-mRNA processing in neurons (3). However, in mammals, only a limited number of splicing regulators have been identified to date, despite the large diversity of the mammalian gene transcripts, and little is known about the mechanisms by which alternative splicing is regulated.Pre-mRNA splicing occurs in a large multicomponent ribonucleoprotein complex called the spliceosome, which is composed of U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein (snRNP) particles and many non-snRNP protein splicing factors (5). U1 snRNP recognizes the 5Ј splice site and is among the first factors to inter...

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently discovered binds to the HTLV-1 provirus. We show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.

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