Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA.

Trim24 (Tif1α) and Trim33 (Tif1γ) interact to form a co-repressor complex that suppresses murine hepatocellular carcinoma. Here we show that Trim24 and Trim33 cooperatively repress retinoic acid receptor-dependent activity of VL30-class endogenous retroviruses (ERVs) in liver. In Trim24-knockout hepatocytes, VL30 derepression leads to accumulation of reverse-transcribed VL30 cDNA in the cytoplasm that correlates with activation of the viral-defense interferon responses mimicking the preneoplastic inflammatory state seen in human liver following exogenous viral infection. Furthermore, upon derepression, VL30 long terminal repeats (LTRs) act as promoter and enhancer elements deregulating expression of neighboring genes and generating enhancer RNAs that are required for LTR enhancer activity in hepatocytes in vivo. These data reinforce the role of the TRIM family of proteins in retroviral restriction and antiviral defense and provide an example of an ERV-derived oncogenic regulatory network.

Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha.

Hepatocellular carcinoma (HCC) is a major cause of death worldwide. Here, we provide evidence that the ligand-dependent nuclear receptor co-regulator Trim24 (also known as Tif1alpha) functions in mice as a liver-specific tumor suppressor. In Trim24-null mice, hepatocytes fail to execute proper cell cycle withdrawal during the neonatal-to-adult transition and continue to cycle in adult livers, becoming prone to a continuum of cellular alterations that progress toward metastatic HCC. Using pharmacological approaches, we show that inhibition of retinoic acid signaling markedly reduces hepatocyte proliferation in Trim24-/- mice. We further show that deletion of a single retinoic acid receptor alpha (Rara) allele in a Trim24-null background suppresses HCC development and restores wild-type expression of retinoic acid-responsive genes in the liver, thus demonstrating that in this genetic background Rara expresses an oncogenic activity correlating with a dysregulation of the retinoic acid signaling pathway. Our results not only provide genetic evidence that Trim24 and Rara co-regulate hepatocarcinogenesis in an antagonistic manner but also suggest that aberrant activation of Rara is deleterious to liver homeostasis.

Dynamic and selective interactions of the transcriptional corepressor TIF1 beta with the heterochromatin protein HP1 isotypes during cell differentiation.

Cell differentiation is a multi-step process marked by progressive silencing of gene expression through mechanisms believed to involve heterochromatin. We have previously shown that interaction between the Krüppel associated box-containing zinc finger proteins (KRAB-ZFP) corepressor TIF1beta and the heterochromatin proteins HP1 is essential for progression through differentiation of embryonal carcinoma F9 cells. This analysis clearly demonstrated the link between gene specific repressors, components of heterochromatin and cell differentiation. In mammals, there are three HP1 isotypes, HP1alpha, beta, and gamma, that appear to be involved in both eu- and heterochromatin, but whose individual functions are still poorly defined. Therefore, the aim of the present study was to determine in vivo (i) which HP1 isotypes interact with TIF1beta, (ii) in which sub-nuclear compartments these interactions occur and (iii) whether these interactions are regulated during cell differentiation. To address these questions, we established stable F9 cell lines co-expressing TIF1beta fused to the ECFP fluorophore and HP1alpha, beta, or gamma fused to the EYFP fluorophore. Using the Föster resonance energy transfer (FRET) technology, we map the physical interaction between TIF1beta-CFP and the different HP1-YFP isotypes in living F9 cells. We demonstrate that in non-differentiated cells, TIF1beta-CFP/HP1-YFP interaction occurs only within euchromatin and involves selectively HP1beta-YFP and HP1gamma-YFP, but not HP1alpha-YFP. Furthermore, in differentiated cells, TIF1beta-CFP selectively associates with HP1beta-YFP within heterochromatin, while TIF1beta-CFP/HP1gamma-YFP is exclusively present within euchromatin. No physical TIF1beta-CFP/HP1alpha-YFP interaction is detected in neither non differentiated nor differentiated cells. These results support the notion that, in vivo, HP1 isotypes have specific nonredundant functions and provide evidence for HP1beta playing an essential role in the shuttling of TIF1beta from eu- to heterochromatin during cell differentiation.

Association of the transcriptional corepressor TIF1beta with heterochromatin protein 1 (HP1): an essential role for progression through differentiation.

The transcriptional intermediary factor 1beta (TIF1beta) is a corepressor for KRAB-domain-containing zinc finger proteins and is believed to play essential roles in cell physiology by regulating chromatin organization at specific loci through association with chromatin remodeling and histone-modifying activities and recruitment of heterochromatin protein 1 (HP1) proteins. In this study, we have engineered a modified embryonal carcinoma F9 cell line (TIF1beta(HP1box/-)) expressing a mutated TIF1beta protein (TIF1beta(HP1box)) unable to interact with HP1 proteins. Phenotypic analysis of TIF1beta(HP1box/-) and TIF1beta(+/-) cells shows that TIF1beta-HP1 interaction is not required for differentiation of F9 cells into primitive endoderm-like (PrE) cells on retinoic acid (RA) treatment but is essential for further differentiation into parietal endoderm-like (PE) cells on addition of cAMP and for differentiation into visceral endoderm-like cells on treatment of vesicles with RA. Complementation experiments reveal that TIF1beta-HP1 interaction is essential only during a short window of time within early differentiating PrE cells to establish a selective transmittable competence to terminally differentiate on further cAMP inducing signal. Moreover, the expression of three endoderm-specific genes, GATA6, HNF4, and Dab2, is down-regulated in TIF1beta(HP1box/-) cells compared with wild-type cells during PrE differentiation. Collectively, these data demonstrate that the interaction between TIF1beta and HP1 proteins is essential for progression through differentiation by regulating the expression of endoderm differentiation master players.

TIF1delta, a novel HP1-interacting member of the transcriptional intermediary factor 1 (TIF1) family expressed by elongating spermatids.

TIF1 (transcriptional intermediary factor 1) proteins are encoded by an expanding family of developmental and physiological control genes that are conserved from flies to man. These proteins are characterized by an N-terminal RING-B box-coiled-coil (RBCC) motif and a C-terminal PHD finger/bromodomain unit, and have been implicated in epigenetic mechanisms of transcriptional repression involving histone modifiers and heterochromatin-binding proteins. We describe here the isolation and functional characterization of a fourth murine TIF1 gene, TIF1delta. The predicted TIF1delta protein displays all the structural hallmarks of a bona fide TIF1 family member and resembles the other TIF1s in that it can exert a deacetylase-dependent silencing effect when tethered to a promoter region. Moreover, like TIF1alpha and TIF1beta, TIF1delta can homodimerize and contains a PXVXL motif necessary and sufficient for HP1 (heterochromatin protein 1) binding. Although TIF1alpha and TIF1beta also bind nuclear receptors and Kruppel-associated boxes specifically and respectively, TIF1delta appears to lack nuclear receptor- and Kruppel-associated box binding activity. Furthermore, TIF1delta is unique among the TIF1 family proteins in that its expression is largely restricted to the testis and confined to haploid elongating spermatids, where it associates preferentially with HP1 isotype gamma (HP1gamma) and forms discrete foci dispersed within the centromeric chromocenter and the surrounding nucleoplasm. Collectively, these data are consistent with specific, nonredundant functions for the TIF1 family members in vivo and suggest a role for TIF1delta in heterochromatin-mediated gene silencing during postmeiotic phases of spermatogenesis.

Molecular cloning, genomic structure, and expression analysis of the mouse transcriptional intermediary factor 1 gamma gene.

Human transcriptional intermediary factor 1 gamma (Tif1gamma), also known as Ret fused gene 7 (RFG7), is a member of a novel family of transcriptional coregulator-encoding genes which function in cell differentiation and development. Here, we report the structure and expression pattern of the mouse Tif1gamma gene. This gene comprises 20 coding exons spanning about 77 kb of genomic DNA on chromosome 3F2, and encodes a 1142-amino-acid protein with 96% identity to the human protein. The locations of exon/intron boundaries correlated well with those for the regions of conserved amino acid sequences (RBCC motif, PHD finger and bromodomain). In situ hybridization analysis of the TIF1gamma mRNA on sections from staged mouse embryos revealed a low level of ubiquitous expression at midgestation, and higher expression levels within the brain and spinal cord epithelium at later developmental stages. Prominent expression was also found in developing sensory epithelia (cochlea, retina, olfactory epithelium), and in several developing organs including the thymus, lung, stomach, intestine, liver, and kidney cortex. In the adult mouse, Tif1gamma mRNA was detected by Northern blot analysis in all tissues examined, with the highest expression level in testis. In situ hybridization and immunohistochemistry studies revealed that expression of the Tif1gamma mRNA and protein varied according to the stage of the seminiferous epithelium cycle. Taken together, these results indicate-and serve as a basis for investigating-a possible involvement of Tif1gamma in the control of embryonic development and spermatogenesis.

Nizp1, a novel multitype zinc finger protein that interacts with the NSD1 histone lysine methyltransferase through a unique C2HR motif.

Haploinsufficiency of the NSD1 gene is a hallmark of Sotos syndrome, and rearrangements of this gene by translocation can cause acute myeloid leukemia. The NSD1 gene product is a SET-domain histone lysine methyltransferase that has previously been shown to interact with nuclear receptors. We describe here a novel NSD1-interacting protein, Nizp1, that contains a SCAN box, a KRAB-A domain, and four consensus C2H2-type zinc fingers preceded by a unique finger derivative, referred to herein as the C2HR motif. The C2HR motif functions to mediate protein-protein interaction with the cysteine-rich (C5HCH) domain of NSD1 in a Zn(II)-dependent fashion, and when tethered to RNA polymerase II promoters, represses transcription in an NSD1-dependent manner. Mutations of the cysteine or histidine residues in the C2HR motif abolish the interaction of Nizp1 with NSD1 and compromise the ability of Nizp1 to repress transcription. Interestingly, converting the C2HR motif into a canonical C2H2 zinc finger has a similar effect. Thus, Nizp1 contains a novel type of zinc finger motif that functions as a docking site for NSD1 and is more than just a degenerate evolutionary remnant of a C2H2 motif.

NSD1 is essential for early post-implantation development and has a catalytically active SET domain.

The nuclear receptor-binding SET domain-containing protein (NSD1) belongs to an emerging family of proteins, which have all been implicated in human malignancy. To gain insight into the biological functions of NSD1, we have generated NSD1-deficient mice by gene disruption. Homozygous mutant NSD1 embryos, which initiate mesoderm formation, display a high incidence of apoptosis and fail to complete gastrulation, indicating that NSD1 is a developmental regulatory protein that exerts function(s) essential for early post-implantation development. We have also examined the enzymatic potential of NSD1 and found that its SET domain possesses intrinsic histone methyltransferase activity with specificity for Lys36 of histone H3 (H3-K36) and Lys20 of histone H4 (H4-K20).

Selective interaction between the chromatin-remodeling factor BRG1 and the heterochromatin-associated protein HP1alpha.

Mammalian heterochromatin protein 1 (HP1) alpha, HP1beta and HP1gamma are closely related non-histone chromosomal proteins that function in gene silencing, presumably by organizing higher order chromatin structures. Here, we show by co-immunoprecipitation that HP1alpha, but neither HP1beta nor HP1gamma, forms a complex with the BRG1 chromatin-remodeling factor in HeLa cells. In vitro, BRG1 interacts directly and preferentially with HP1alpha. The region conferring this preferential binding has been mapped to residues 106-180 of the HP1alpha C-terminal chromoshadow domain. Using site-directed mutagenesis, we have identified three amino acid residues I113, A114 and C133 in HP1alpha (K, P and S in HP1beta and HP1gamma) that are essential for the selective interaction of HP1alpha with BRG1. Interestingly, these residues were also shown to be critical for the silencing activity of HP1alpha. Taken together, these results demonstrate that mammalian HP1 proteins are biochemically distinct and suggest an entirely novel function for BRG1 in modulating HP1alpha-containing heterochromatic structures.

Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells.

The myotubularin-related 1 (MTMR1) gene belongs to a highly conserved family of eucaryotic phosphatases, with at least 11 members in humans. The founder member of this gene family, MTM1, is mutated in X-linked myotubular myopathy, a severe congenital disorder that affects skeletal muscle, and codes for myotubularin, a specific phosphatidylinositol 3-phosphate [PI(3)P] phosphatase. MTM1 and MTMR1 are adjacent on the X chromosome, and the corresponding proteins share 59% sequence identity. In the present study, we investigated the putative role of MTMR1 in myogenesis by analysing its expression pattern in muscle cells during differentiation and in skeletal muscle throughout development. We have identified three novel coding exons in the MTMR1 intron 2 that are conserved between mouse and human, are alternatively spliced, and give rise to six mRNA isoforms. One of the transcripts is muscle-specific and is induced during myogenesis both in vitro and in vivo, and represents the major isoform in adult skeletal muscle. We show that the two main MTMR1 protein muscular isoforms, like myotubularin, efficiently dephosphorylate PI(3)P in vitro. We have also analysed whether MTMR1 alternative splicing is affected in skeletal muscle cells derived from patients with congenital myotonic dystrophy (cDM1), in which mRNA splicing disturbances of specific genes are thought to constitute an important pathogenic mechanism. We found a striking reduction in the level of the muscle-specific isoform and the appearance of an abnormal MTMR1 transcript in differentiated cDM1 muscle cells in culture and in skeletal muscle from cDM1 patients. Our results suggest that MTMR1 plays a role in muscle formation and represents a novel target for abnormal mRNA splicing in myotonic dystrophy.