Giovanna Grimaldi

In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions.

Summary The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.

A variety of RNA polymerases II and III-dependent promoter classes is repressed by factors containing the Krüppel-associated/finger preceding box of zinc finger proteins.

KRAB/FPB (Krüppel-associated/finger preceding box) domains are small, portable transcriptional repression motifs, encoded by hundreds of vertebrates C2-H2-type zinc finger genes. We report that KRAB/FPB domains feature an unprecedented, highly promiscuous DNA-binding dependent transcriptional repressing activity. Indeed, template bound chimeric factors containing KRAB/FPB modules actively repress in vivo the transcription of distinct promoter classes that depend on different core elements, recruit distinct basal transcriptional apparatuses and are transcribed either by RNA polymerase II or III. The promoter types repressed in transient assays in a dose- and DNA-binding dependent, but position- and orientation-independent manner, by GAL4-KRAB/FPB fusions include an RNA polymerase II-dependent small nuclear RNA promoter (U1) as well as RNA polymerase III-dependent class 2 (adenovirus VA1), class 3 (human U6) and atypical (human 7SL) promoters. Down-modulation of all of these templates depended on factors containing the A module of the KRAB/FPB domain. Data provide further insights into the properties and mode of action of this widespread repression motif, and support the notion that genes belonging to distinct classes may be repressed in vivo by KRAB/FPB containing zinc finger proteins. The exquisitely DNA-binding dependent transcriptional promiscuity exhibited by KRAB/FPB domains may provide a unique model system for studying the mechanism by which a promoter recruited repression motif can down-modulate a large variety of promoter types.

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.

Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1

In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGCmetCGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.

Coding region intron/exon organization, alternative splicing, and X-chromosome inactivation of the KRAB/FPB-domain-containing human zinc finger gene ZNF41

ZNF41 belongs to a cluster of human zinc finger genes residing within a gene-rich region at Xp11.23. ZNF41 encodes a KRAB/FPB (Krüppel-associated/finger preceding box) domain, a potent transcription repression motif present in hundreds of vertebrate zinc finger protein genes, composed of two protein modules, A and B. Three introns, placed at identical positions in paralogous genes, interrupt four exons encoding the ZNF41 N-terminal amino acids, the KRAB/FPB-A and KRAB/FPB-B modules, and the remaining coding region adjoined to the C-terminal zinc finger domain. Since the KRAB/FPB-A and KRAB/FPB-B modules are encoded by dedicated exons in ZNF41 and paralogous genes, exon skipping may lead to differential usage of these modules in alternative gene products. RT-PCR analysis of ZNF41 mRNAs showed that, while skipping of the KRAB/FPB-A and/or KRAB/FPB-B exons was not detected, the use of alternative donor/acceptor sites upstream of the KRAB/FPB-A exon generates multiple ZNF41 transcripts potentially encoding polypeptides differing in the N-terminal region and expressed in different tissues. The expression pattern in cell hybrids containing either active or inactive X chromosomes indicates that ZNF41, which resides within a region of the X chromosome that includes genes that are both subject to and escape X-inactivation, is susceptible to X-chromosome inactivation.

Isolation and expression analysis of a human zinc finger gene (ZNF41) located on the short arm of the X chromosome

We have isolated a novel human zinc finger gene, ZNF41, from a human X-chromosome-specific library. Nucleotide sequence analysis reveals that ZNF41 potentially encodes a polypeptide featuring an array of 18 contiguous zinc fingers of the C2H2 type. Multiple polyadenylated transcripts homologous to ZNF41 are present at different levels in several distinct cell types. Southern analyses of somatic cell hybrids containing either intact or rearranged X chromosomes confirm the genomic origin of the isolated gene and establish that it is localized between Xcen and Xp22.1.

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

Fungal extrachromosomal DNA and its maintenance and expression in E. coli K-12

A phytopathogenic fungus, Fusarium oxysporum , has been found to contain a plasmid-like DNA molecule which can be introduced by transformation into E. coli K-12 strains, where it can be maintained as a bacteria plasmid. An E. coli K-12 strain bearing this fungal DNA is able to express a polysaccharide hydrolizing activity which is known to be involved in the fungal pathogenic processes.

A novel X-linked member of the human zinc finger protein gene family: isolation, mapping, and expression

We report the partial characterization of a novel putative zinc finger gene of the Krüppel-type (ZNF81), isolated from an X Chromosome (Chr) specific library. The pattern of segregation in human-hamster somatic cell hybrids of sequences homologous to the ZNF81 finger domain has established that it resides within the Xp22.1-Xp11 region. ZNF81 represents yet another example, together with ZFX, ZNF41, and ZNF21, of members of the zinc finger gene family residing within the short arm of the human X Chr. Sequence analysis showed that ZNF81 may encode a polypeptide(s) containing tandem arrays of 12 canonical C2H2 zinc fingers of the Krüppel-type at the C-terminus. Northern analysis indicated that probes from the ZNF81 finger domain hybridize to polyadenylated transcripts present in several cell lines, a result that supports the hypothesis that it is an expressed, functional member of this multigene family.