PubMed Health⌕ Search

Biomedical subjects

Frank J Rauscher

Publications and source records attributed to Frank J Rauscher.

9 recordsLinked to original sources
PubMed2006

KAP1, a novel substrate for PIKK family members, colocalizes with numerous damage response factors at DNA lesions.

The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events, which ultimately converge on damaged DNA packaged in chromatin. Few connections between the proteins that remodel chromatin and the proteins that mediate this damage response have been shown. We have investigated the DNA damage-induced phosphorylation of the KRAB-ZFP-associated protein 1 (KAP1), the dedicated corepressor for Krüppel-associated box (KRAB) zinc finger protein (ZFP) proteins. We show that KAP1 is rapidly phosphorylated following DNA damage by members of the phosphatidylinositol-3 kinase-like family of kinases. This phosphorylation occurs at a single amino acid residue that is conserved from mice to humans and is located adjacent to the bromodomain, suggesting that it may regulate chromatin recognition by that module. Phosphorylated KAP1 rapidly localizes to sites of DNA strand breaks in the nucleus in response to ionizing radiation. This discovery provides a novel link between chromatin-mediated transcriptional repression and the recognition/repair of DNA, which must be accomplished by the cellular DNA damage response.

Cell Division↗
PubMed2005

MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation.

MDM2 is a RING domain ubiquitin E3 ligase and a major regulator of the p53 tumor suppressor. MDM2 binds to p53, inactivates p53 transcription function, inhibits p53 acetylation, and promotes p53 degradation. Here, we present evidence that MDM2 interacts with the nuclear corepressor KAP1. The binding is mediated by the N-terminal coiled-coil domain of KAP1 and the central acidic domain of MDM2. KAP1 stimulates formation of p53-HDAC1 complex and inhibits p53 acetylation by interacting with MDM2. Expression of KAP1 cooperates with MDM2 to promote p53 ubiquitination and degradation. The tumor suppressor ARF competes with KAP1 in MDM2 binding; oncogene induction of ARF expression reduces MDM2-KAP1 interaction. Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis. Therefore, MDM2 interaction with KAP1 contributes to p53 functional regulation. ARF may regulate p53 acetylation and stability in part by inhibiting KAP1-MDM2 binding.

Acetylation↗
PubMed2005

The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain.

The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

Amino Acid Motifs↗
PubMed2003

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation.

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.

3T3 Cells↗
PubMed2003

Novel consensus DNA-binding sequence for BRCA1 protein complexes.

Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA-binding complex. Interestingly, these DNA-binding complexes are downregulated in breast cancer cell lines containing wild-type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA-binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA-binding sequence and indicate that one function of BRCA1 is to co-regulate the expression of genes involved in various cellular processes.

BRCA1 Protein↗
PubMed2002

Hetero-oligomerization among the TIF family of RBCC/TRIM domain-containing nuclear cofactors: a potential mechanism for regulating the switch between coactivation and corepression.

The RING-B box-coiled-coil (RBCC) motif (also re-named recently as the tripartite motif (TRIM)) is a widely distributed motif that is hypothesized to be a protein-protein interface. The RBCC/TRIM domain of the corepressor KAP-1 is both necessary and sufficient to interact directly with the transcription repressor KRAB domain. Each subdomain of the KAP-1-RBCC contributes directly to the oligomerization and/or ligand recognition. Little is known about the function or the natural binding ligands for the RBCC/TRIM domain of the other TIF family members. In order to investigate whether hetero-oligomerization might be a biological regulatory mechanism, we have evaluated the hetero-oligomerization potential of the TIF family members including KAP-1, TIF1alpha, TIF1gamma, and the RBCC/TRIM family members including PML1, and MID1. We have reconstituted and characterized the oligomerization for these proteins using baculovirus and mammalian expression systems by biochemical approaches. Our data indicate that the RBCC/TRIM domains of KAP-1, TIF1alpha and TIF1gamma exist in a homo-oligomeric state. However, there is little cross-talk between KAP-1 and other TIF family members, suggesting that a high degree of specificity for oligomerization interface and ligand recognition is intrinsically built into the RBCC/TRIM domain of KAP-1. Finally, we demonstrate that TIF1alpha interacts with TIF1gamma and the coiled-coil region of TIF1gamma is necessary for this interaction. The hetero-oligomerization between TIF1alpha and TIF1gamma implies a potential regulatory mechanism for transcriptional regulation.

Animals↗
PubMed2002

A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.

More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

3T3 Cells↗
PubMed2002

SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins.

Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. Here we report that the KAP-1 corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, we found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localized SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression.

Animals↗