Stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha) transcription complex, caused by intratumoural hypoxia, promotes tumour progression and metastasis, leading to treatment failure and mortality in different types of human cancers. The transcription factor TWIST is a master regulator of gastrulation and mesoderm-specification and was implicated recently as an essential mediator of cancer metastasis. Notably, HIF-1alpha- and TWIST-null mice show similarities in their phenotypes. Here, we have shown that hypoxia or overexpression of HIF-1alpha promotes epithelial-mesenchymal transition (EMT) and metastastic phenotypes. We also found that HIF-1 regulates the expression of TWIST by binding directly to the hypoxia-response element (HRE) in the TWIST proximal promoter. However, siRNA-mediated repression of TWIST in HIF-1alpha-overexpressing or hypoxic cells reversed EMT and metastastic phenotypes. Co-expression of HIF-1alpha, TWIST and Snail in primary tumours of patients with head and neck cancers correlated with metastasis and the worst prognosis. These results provide evidence of a key signalling pathway involving HIF-1alpha and TWIST that promotes metastasis in response to intratumoural hypoxia.

The CUL4-DDB1-ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4-DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4-DDB1 (ref. 8). We found that CUL4A-DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4-DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.

A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. Set1/COMPASS was the founding member and is the only H3K4 methylase in Saccharomyces cerevisiae; however, in mammals, at least six H3K4 methylases, Set1A and Set1B and MLL1 to MLL4, are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation patterns in wild-type Mll1 ؉/؉ and Mll1 ؊/؊ mouse embryonic fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes, show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/MLL4 and/or the Set1 complexes has little to no effect on the H3K4 methylation of Hox loci or their expression levels in these MEFs. Together these data provide insight into the redundancy and specialization of COMPASS-like complexes in mammals and provide evidence for a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation.The mixed-lineage leukemia gene (MLL1) is involved in numerous translocations found in several human acute leukemias (2,43,53). Chromosomal translocations in the MLL1 gene result in hematological malignancies, including acute myeloid and lymphoid leukemia. Although these cytogenetic abnormalities were discovered over 25 years ago, little is known about the biochemical functions of MLL1, its protein complexes, its translocation partners, and particularly, why these translocations result in leukemia. MLL1 is one of at least six genes encoding histone methyltransferases in mammals that methylate histone H3 on lysine 4 (H3K4), a posttranslational mark primarily associated with promoters of active genes (49). Much of our knowledge of the implementation of this mark comes from studies of Set1/COMPASS in the yeast Saccharomyces cerevisiae. The first H3K4 methylase complex to be identified, COMPASS, was purified from yeast and contains Set1 and seven other polypeptides, named Cps60 to Cps15 according to their size in yeast (30). COMPASS is capable of mono-, di-, and trimethylating H3K4 (20,42,48,56). Subsequently, it was determined that six mammalian Set1 homologs, MLL1 to MLL4 (KMT2A to KMT2D) and human Set1A and human Set1B (KMT2F and KMT2G), are found in COMPASS-like complexes, all capable of methylating H3K4 (5,15,16,23,38,49,59).We have recently shown that human Set1A/Set1B function most similarly to yeast COMPASS and mediate the bulk of the H3K4 trimethylation in mammalian cell extracts (57). In contrast, the members of th...

In yeast, the macromolecular complex Set1/COMPASS is capable of methylating H3K4, a posttranslational modification associated with actively transcribed genes. There is only one Set1 in yeast; yet in mammalian cells there are multiple H3K4 methylases, including Set1A/B, forming human COMPASS complexes, and MLL1-4, forming human COMPASS-like complexes. We have shown that Wdr82, which associates with chromatin in a histone H2B ubiquitination-dependent manner, is a specific component of Set1 complexes but not that of MLL1-4 complexes. RNA interference-mediated knockdown of Wdr82 results in a reduction in the H3K4 trimethylation levels, although these cells still possess active MLL complexes. Comprehensive in vitro enzymatic studies with Set1 and MLL complexes demonstrated that the Set1 complex is a more robust H3K4 trimethylase in vitro than the MLL complexes. Given our in vivo and in vitro observations, it appears that the human Set1 complex plays a more widespread role in H3K4 trimethylation than do the MLL complexes in mammalian cells.The MLL gene located on chromosome 11q23 is found in a variety of chromosomal translocations possessing unique clinical and biological characteristics (11,23,31). The MLL rearrangements are found in more than 70% of infant leukemia, with phenotypes more consistent with acute lymphoid leukemia and/or acute myeloid leukemia (23). Such translocations have also been reported in ca. 10% of adults with a diagnosis of acute myeloid leukemia related to the treatment of other malignancies with the use of topoisomerase II inhibitors.For more than 20 years, little was known about the molecular function(s) of MLL until its yeast homologue, Set1, was identified in a macromolecular complex named COMPASS (for complex of proteins associated with Set1) (18). COM-PASS is capable of mono-, di-, and trimethylating histone H3 on lysine 4 (H3K4) (12,18,20,22,26). We now know that human MLL is also found in a COMPASS-like complex capable of methylating H3K4 (7,35). In addition to MLL, there are three MLL homologues (MLL2 to MLL4) (3, 7) and two Set1-related proteins (Set1A and Set1B) (13, 15) in humans; all are found in COMPASS-like complexes capable of methylating H3K4 (26, 27). We do not understand why there are several H3K4 methylases in mammalian cells capable of methylating H3K4. However, it is clear that the H3K4 methylase activities in mammals are not redundant. This is evident by the observation that the deletion of many individual MLL genes result in embryonic lethality (6, 36). Why do mammals need so many different H3K4 methylases? Perhaps mammals need to control the H3K27 methylation mark, which is associated with polycomb and transcriptional silencing at different genomic loci in different cellular contexts, while single-celled eukaryotes do not (1). It is possible that mammals have a built-in intricate network of H3K4 methylases that perform the ancient functions, as well as oppose and perhaps reverse H3K27. All of the H3K4 methylase-containing complexes in mammals have COMPASS-like conserved...

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

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Epithelial-mesenchymal transition (EMT) is important for organ development, metastasis, cancer stemness, and organ fibrosis. Molecular mechanisms to coordinately regulate hypoxia-induced EMT remain elusive. Here, we show that HIF-1α-induced histone deacetylase 3 (hdac3) is essential for hypoxia-induced EMT and metastatic phenotypes. Change of specific chromatin states is associated with hypoxia-induced EMT. Under hypoxia, HDAC3 interacts with hypoxia-induced WDR5, recruits the histone methyltransferase (HMT) complex to increase histone H3 lysine 4 (H3K4)-specific HMT activity, and activates mesenchymal gene expression. HDAC3 also serves as an essential corepressor to repress epithelial gene expression. Knockdown of WDR5 abolishes mesenchymal gene activation but not epithelial gene repression during hypoxia. These results indicate that hypoxia induces different chromatin modifiers to coordinately regulate EMT through distinct mechanisms.

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