NORMAL GENOMIC

• Fleischer S, Wiemann S, Will H, Hofmann TG
• PML-associated repressor of transcription (PAROT), a novel KRAB-zincfinger repressor, is regulated through association with PML nuclearbodies.
• Exp Cell Res. 2006; 312: 901-12
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• Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated intranscriptional regulation. Here we identify a novel transcriptionalrepressor, PML-associated repressor of transcription (PAROT), which isregulated in its repressor activity through recruitment to PML-NBs. PAROTis a Kruppel-associated box ( KRAB) zinc-finger (ZNF) protein, whichcomprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8tandemly repeated C(2)H(2)-ZNF motifs at its carboxy terminus. Consistentwith its domain structure, when tethered to DNA, PAROT repressestranscription, and this is partially released by the HDAC inhibitortrichostatin A. PAROT colocalizes with members of the heterochromatinprotein 1 (HP1) family and with transcriptional intermediaryfactor-1beta/KRAB-associated protein 1 (TIF-1beta/KAP1), a transcriptionalcorepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, incontrast to PML-III, efficiently recruits PAROT and TIF-1beta fromheterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releasesits transcriptional repressor activity, indicating that PAROT can beregulated through subnuclear compartmentalization. Taken together, ourdata identify a novel transcriptional repressor and provide evidence forits regulation through association with PML-NBs.

• Shannon M, Hamilton AT, Gordon L, Branscomb E, Stubbs L
• Differential expansion of zinc-finger transcription factor loci inhomologous human and mouse gene clusters.
• Genome Res. 2003; 13: 1097-110
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• Mammalian genomes carry hundreds of Kruppel-type zinc finger (ZNF) genes,most of which reside in familial clusters. ZNF genes encodingKruppel-associated box (KRAB) motifs are especially prone to this type oftandem organization. Despite their prevalence, little is known about thefunctions or evolutionary histories of these clustered gene families. Herewe describe a homologous pair of human and mouse KRAB-ZNF gene clusterscontaining 21 human and 10 mouse genes, respectively. Evolutionaryanalysis uncovered only three pairs of putative orthologs and two caseswhere a single gene in one species is related to multiple genes in theother; several human genes have no obvious homolog in mouse. We deducethat duplication and loss of ancestral cluster members occurredindependently in the primate and rodent lineages after divergence,yielding substantially different ZNF gene repertoires in humans and mice.Differences in expression patterns and sequence divergence within the DNAbinding regions of predicted proteins suggest that the duplicated geneshave acquired novel functions over evolutionary time. Since KRAB-ZNFproteins are predicted to function as transcriptional regulators, theelaboration of new lineage-specific genes in this and other clustered ZNFfamilies is likely to have had a significant impact on species-specificaspects of biology.

• Venturini L et al.
• TIF1gamma, a novel member of the transcriptional intermediary factor 1family.
• Oncogene. 1999; 18: 1209-17
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• We report the cloning and characterization of a novel member of theTranscriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma.Similar to TIF1alpha and TIF1beta, the structure of TIF1beta ischaracterized by multiple domains: RING finger, B boxes, Coiled coil,PHD/TTC, and bromodomain. Although structurally related to TIF1alpha andTIF1beta, TIF1gamma presents several functional differences. In contrastto TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclearreceptors in yeast two-hybrid or GST pull-down assays and does notinterfere with retinoic acid response in transfected mammalian cells.Whereas TIF1alpha and TIF1beta were previously found to interact with theKRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) andMOD2 (HP1gamma) heterochromatinic proteins, suggesting that they mayparticipate in a complex involved in heterochromatin-induced generepression, TIF1gamma does not interact with either the KRAB domain ofKOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha andTIF1beta, exhibits a strong silencing activity when tethered to apromoter. Since deletion of a novel motif unique to the three TIF1proteins, called TIF1 signature sequence (TSS), abrogates transcriptionalrepression by TIF1gamma, this motif likely participates in TIF1 dependentrepression.

• Elser B, Kriz W, Bonventre JV, Englert C, Witzgall R
• The Kruppel-associated box (KRAB)-zinc finger protein Kid-1 and the Wilms'tumor protein WT1, two transcriptional repressor proteins, bind toheteroduplex DNA.
• J Biol Chem. 1997; 272: 27908-12
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• Zinc finger proteins of the Cys2His2 class represent a large group ofDNA-binding proteins. A major subfamily of those proteins, theKruppel-associated box (KRAB) domain-containing Cys2His2-zinc fingerproteins, have been described as potent transcriptional repressors. Sofar, however, no DNA-binding sites for KRAB domain-containing zinc fingerproteins have been isolated. Using a polymerase chain reaction-basedselection strategy with double- and single-stranded DNA, we failed toreveal a binding site for Kid-1, one member of KRAB-zinc finger proteins.Binding of Kid-1 both to single- and homoduplex double-stranded DNA wasnegligible. We now present evidence that Kid-1 binds to heteroduplex DNA.Similar to Kid-1, the non-KRAB-zinc finger protein WT1 also bound avidlyto heteroduplex DNA (both the -KTS and +KTS splice variant of WT1),whereas the POU domain protein Oct-6, the ets domain protein Ets-1 and theRING finger of BRCA-1 did not bind to heteroduplex DNA. Binding of WT1 toheteroduplex DNA was markedly reduced in naturally occurring mutants. Therecognition of certain DNA structures by transcriptional repressorproteins may therefore represent a more common phenomenon than previouslythought.

• Ayer DE, Laherty CD, Lawrence QA, Armstrong AP, Eisenman RN
• Mad proteins contain a dominant transcription repression domain.
• Mol Cell Biol. 1996; 16: 5772-81
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• Transcription repression by the basic region-helix-loop-helix-zipper(bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Maxand mSin3A or mSin3B, the mammalian orthologs of the Saccharomycescerevisiae transcriptional corepressor SIN3. The interaction between Mad1and mSin3 is mediated by three potential amphipathic alpha-helices: one inthe N terminus of Mad (mSin interaction domain, or SID) and two within thesecond paired amphipathic helix domain (PAH2) of mSin3A. Mutations thatalter the structure of the SID inhibit in vitro interaction between Madand mSin3 and inactivate Mad's transcriptional repression activity. Herewe show that a 35-residue region containing the SID represents a dominantrepression domain whose activity can be transferred to a heterologous DNAbinding region. A fusion protein comprising the Mad1 SID linked to a Ga14DNA binding domain mediates repression of minimal as well as complexpromoters dependent on Ga14 DNA binding sites. In addition, the SIDrepresses the transcriptional activity of linked VP16 and c-Myctransactivation domains. When fused to a full-length c-Myc protein, theMad1 SID specifically represses both c-Myc's transcriptional andtransforming activities. Fusions between the GAL DNA binding domain andfull-length mSin3 were also capable of repression. We show that theassociation between Mad1 and mSin3 is not only dependent on the helicalSID but is also dependent on both putative helices of the mSin3 PAH2region, suggesting that stable interaction requires all three helices. Ourresults indicate that the SID is necessary and sufficient fortranscriptional repression mediated by the Mad protein family and that SIDrepression is dominant over several distinct transcriptional activators.

• Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher FJ 3rd
• Kruppel-associated boxes are potent transcriptional repression domains.
• Proc Natl Acad Sci U S A. 1994; 91: 4509-13
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• The Kruppel-associated box (KRAB) is a highly conserved, 75-aa regioncontaining two predicted amphipathic alpha-helices. The KRAB domain ispresent in the amino-terminal regions of more than one-third of allKruppel-class Cys2His2 zinc finger proteins and is conserved from yeast toman; however, its function is unknown. Here it is shown that the KRABdomain functions as a DNA binding-dependent transcriptional repressor whenfused to a heterologous DNA-binding domain from the yeast GAL4 protein. A45-aa segment containing one of the predicted KRAB amphipathic helices wasnecessary and sufficient for repression. Amino acid substitutions in thepredicted helix abolished the repression function. These results assign afunction, transcriptional repression, to the highly conserved KRAB box anddefine a minimal repression domain which may aid in identifying mechanismsof repression.

• Constantinou-Deltas CD, Gilbert J, Bartlett RJ, Herbstreith M, Roses AD, Lee JE
• The identification and characterization of KRAB-domain-containing zincfinger proteins.
• Genomics. 1992; 12: 581-9
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• The zinc finger motif is a highly conserved tandemly repeated sequence of28-30 amino acids that was first identified in transcription factor TFIIIAfrom Xenopus laevis. Subsequently, similar motifs were found andcharacterized in many genes from mammalian genomes and the genomes oflower eukaryotes such as Drosophila and yeast, thereby defining a largesuperfamily of genes. Non-finger-coding modules conserved among members ofsubfamilies of zinc finger genes have been described in the murine genome(finger-associated boxes, or FAX domain) and the human genome(Kruppel-associated boxes, or KRAB domain). Here we report theidentification and partial characterization of more members of the humanKRAB-containing subfamily of genes. Based on Southern blot hybridizationexperiments, they also are zinc-finger-coding genes. All members share ahighly homologous 42-amino-acid-long A element of the described KRABdomain. The conservation extends to the murine developmentally expressedzinc finger gene, mKr2. The homologous sequences, however, are part of the5'-untranslated region. In all cases for which there is adequateinformation, the KRAB domain is found at the NH2-terminus of therespective protein. In one zinc-finger-encoding cDNA clone that wecharacterized further in this work, BRc1744 (ZNF45), the KRAB domain mostprobably constitutes the entire second exon of the gene. Based on thedata, it is tempting to speculate that the FAX- and KRAB-containing zincfinger genes define subfamilies of genes with overlapping functions thatparticipate in the regulation of common or similar developmental programs.

• Estruch F
• The yeast putative transcriptional repressor RGM1 is a proline-rich zincfinger protein.
• Nucleic Acids Res. 1991; 19: 4873-7
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• I have cloned a yeast gene, RGM1, which encodes a proline-rich zinc,finger protein. rgm1 mutants do not show any obvious phenotype butoverexpression of RGM1 gene greatly impairs cell growth. The proline-richregion of RGM1 attached to a heterologous DNA binding domain is able torepress the expression of the target gene. RGM1 shares similar zinc fingermotifs with the mammalian Egr (early growth response) proteins as well asproline-rich sequences with a high serine and threonine content,suggesting that RGM1 and Egr proteins could have functional similarities.