Joomyeong Kim

AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2

AEBP2 is a zinc finger protein that has been shown to interact with the mammalian Polycomb Repression Complex 2 (PRC2). In the current study, we characterized this unknown protein and tested its potential targeting roles for the PRC2. AEBP2 is an evolutionarily well-conserved gene that is found in the animals ranging from flying insects to mammals. The transcription of mammalian AEBP2 is driven by two alternative promoters and produces at least two isoforms of the protein. These isoforms show developmental stage-specific expression patterns: the adult-specific larger form (51 kDa) and the embryo-specific smaller form (32 kDa). The AEBP2 protein binds to a DNA-binding motif with an unusual bipartite structure, CTT(N)15-23cagGCC with lower-case being less critical. A large fraction of AEBP2s target loci also map closely to the known target loci of the PRC2. In fact, many of these loci are co-occupied by the two proteins, AEBP2 and SUZ12. This suggests that AEBP2 is most likely a targeting protein for the mammalian PRC2 complex.

Retroposition and evolution of the DNA-binding motifs of YY1, YY2 and REX1

YY1 is a DNA-binding transcription factor found in both vertebrates and invertebrates. Database searches identified 62 YY1 related sequences from all the available genome sequences ranging from flying insects to human. These sequences are characterized by high levels of sequence conservation, ranging from 66% to 100% similarity, in the zinc finger DNA-binding domain of the predicted proteins. Phylogenetic analyses uncovered duplication events of YY1 in several different lineages, including flies, fish and mammals. Retroposition is responsible for generating one duplicate in flies, PHOL from PHO, and two duplicates in placental mammals, YY2 and Reduced Expression 1 (REX1) from YY1. DNA-binding motif studies have demonstrated that YY2 still binds to the same consensus sequence as YY1 but with much lower affinity. In contrast, REX1 binds to DNA motifs divergent from YY1, but the binding motifs of REX1 and YY1 share some similarity at their core regions (5′-CCAT-3′). This suggests that the two duplicates, YY2 and REX1, although generated through similar retroposition events have undergone different selection schemes to adapt to new roles in placental mammals. Overall, the conservation of YY2 and REX1 in all placental mammals predicts that each duplicate has co-evolved with some unique features of eutherian mammals.

SPORADIC AMPLIFICATION OF ID ELEMENTS IN RODENTS

ID sequences are members of a short interspersed element (SINE) repetitive DNA family within the rodent genome. The copy number of individual ID elements varies by up to three orders of magnitude between species. This amplification has been highly sporadic in the order Rodentia and does not follow any phylogenetic trend. Using library screening and dot-blot analysis, we estimate there are 25,000 copies of ID elements in the deer mouse, 1,500 copies in the gerbil (both cricetid rodents), and 60,000 copies of either ID or ID-like elements in a sciurid rodent (squirrel). By dot-blot analysis, we estimate there are 150,000, 4,000, 1,000, and 200 copies of ID elements in the rat, mouse, hamster, and guinea pig, respectively (which is consistent with previous reports) and 200 copies in the hystricognath rodent, nutria. Therefore, a rapid amplification took place not only after the divergence of rat and mouse but also following the deer mouse (Peromyscus) and hamster split, with no evidence of increased amplifications in hystricognath rodents. No notable variations of sequences from the BC1 genes of several myomorphic rodents were observed that would possibly explain the varied levels of ID amplification. We did observe subgenera and species-group-specific variation in the ID core sequence of the BC1 gene within the genus Peromyscus. Sequence analysis of cloned ID elements in Peromyscus show most ID elements in this genus arose prior to Peromyscus subgenus divergence. Correspondence of the consensus sequence of individual ID elements in gerbil and deer mouse further confirms BC1 as a master gene in ID amplification. Several possible mechanisms responsible for the quantitative variations are explored.

Peg3 Mutational Effects on Reproduction and Placenta-Specific Gene Families

Peg3 (paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein. This gene plays important roles in controlling fetal growth rates and nurturing behaviors. In the current study, a new mutant mouse model has been generated to further characterize the functions of this DNA-binding protein. Besides known phenotypes, this new mutant model also revealed potential roles of Peg3 in mammalian reproduction. Female heterozygotes produce a much smaller number of mature oocytes than the wild-type littermates, resulting in reduced litter sizes. According to genome-wide expression analyses, several placenta-specific gene families are de-repressed in the brain of Peg3 heterozygous embryos, including prolactin, cathepsin and carcinoembryonic antigen cell adhesion molecule (Ceacam) families. The observed de-repression is more pronounced in females than in males. The de-repression of several members of these gene families is observed even in the adult brain, suggesting potential defects in epigenetic setting of the placenta-specific gene families in the Peg3 mutants. Overall, these results indicate that Peg3 likely controls the transcription of several placenta-specific gene families, and further suggest that this predicted transcriptional control by Peg3 might be mediated through unknown epigenetic mechanisms.

DNA-binding motif and target genes of the imprinted transcription factor PEG3

The Peg3 gene is expressed only from the paternally inherited allele located on proximal mouse chromosome 7. The PEG3 protein encoded by this imprinted gene is predicted to bind DNA based on its multiple zinc finger motifs and nuclear localization. In the current study, we demonstrated PEG3s DNA-binding ability by characterizing its binding motif and target genes. We successfully identified target regions bound by PEG3 from mouse brain extracts using chromatin immunoprecipitation analysis. PEG3 was demonstrated to bind these candidate regions through the consensus DNA-binding motif AGTnnCnnnTGGCT. In vitro promoter assays established that PEG3 controls the expression of a given gene through this motif. Consistent with these observations, the transcriptional levels of a subset of the target genes are also affected in a mutant mouse model with reduced levels of PEG3 protein. Overall, these results confirm PEG3 as a DNA-binding protein controlling specific target genes that are involved in distinct cellular functions.

DNA methylation analysis of the mammalian PEG3 imprinted domain.

In this study, we performed the first systematic survey of DNA methylation status of the CpG islands of the PEG3 (Paternally expressed gene 3) imprinted domain in the mouse, cow, and human genomes. Previous studies have shown that the region surrounding the first exon of PEG3 contains a differentially methylated CpG island. In addition, we have discovered two previously unreported differentially methylated regions (DMR): one in the promoter region of mouse Zim3 and another in the promoter region of human USP29. In the cow, the Peg3-CpG island was the only area that showed DMR status. We have also examined the methylation status of several CpG islands in this region using human tumor-derived DNA. The CpG islands near PEG3 and USP29 both showed hypermethylation in DNA derived from breast and ovarian tumors. Overall, this study shows that the PEG3 imprinted domain of humans, cows, and mice contains differing numbers of DMRs, but the PEG3-CpG island is the only DMR that is conserved among these three species.

Transcription factor YY1 is essential for regulation of the Th2 cytokine locus and for Th2 cell differentiation

The Th2 locus control region (LCR) has been shown to be important in efficient and coordinated cytokine gene regulation during Th2 cell differentiation. However, the molecular mechanism for this is poorly understood. To study the molecular mechanism of the Th2 LCR, we searched for proteins binding to it. We discovered that transcription factor YY1 bound to the LCR and the entire Th2 cytokine locus in a Th2-specific manner. Retroviral overexpression of YY1 induced Th2 cytokine expression. CD4-specific knockdown of YY1 in mice caused marked reduction in Th2 cytokine expression, repressed chromatin remodeling, decreased intrachromosomal interactions, and resistance in an animal model of asthma. YY1 physically associated with GATA-binding protein-3 (GATA3) and is required for GATA3 binding to the locus. YY1 bound to the regulatory elements in the locus before GATA3 binding. Thus, YY1 cooperates with GATA3 and is required for regulation of the Th2 cytokine locus and Th2 cell differentiation.

Imprinting control region (ICR) of the Peg3 domain

The imprinting and transcription of the 500 kb genomic region surrounding the mouse Peg3 is predicted to be regulated by the Peg3-differentially methylated region (DMR). In the current study, this prediction was tested using a mutant mouse line lacking this potential imprinting control region (ICR). At the organismal level, paternal and maternal transmission of this knockout (KO) allele caused either reduced or increased growth rates in the mouse, respectively. In terms of the imprinting control, the paternal transmission of the KO allele resulted in bi-allelic expression of the normally maternally expressed Zim2, whereas the maternal transmission switched the transcriptionally dominant allele for Zfp264 (paternal to maternal). However, the allele-specific DNA methylation patterns of the DMRs of Peg3, Zim2 and Zim3 were not affected in the mice that inherited the KO allele either paternally or maternally. In terms of the transcriptional control, the paternal transmission caused a dramatic down-regulation in Peg3 expression, but overall up-regulation in the other nearby imprinted genes. Taken together, deletion of the Peg3-DMR caused global changes in the imprinting and transcription of the Peg3 domain, confirming that the Peg3-DMR is an ICR for this imprinted domain.

Rex1/Zfp42 as an epigenetic regulator for genomic imprinting

Zfp42/Rex1 (reduced expression gene 1) is a well-known stem-cell marker that has been duplicated from YY1 in the eutherian lineage. In the current study, we characterized the in vivo roles of Rex1 using a mutant mouse line disrupting its transcription. In contrast to the ubiquitous expression of YY1, Rex1 is expressed only during spermatogenesis and early embryogenesis and also in a very limited area of the placenta. Yet, the gene dosage of Rex1 is very critical for the survival of the late-stage embryos and neonates. This delayed phenotypic consequence suggests potential roles for Rex1 in establishing and maintaining unknown epigenetic modifications. Consistently, Rex1-null blastocysts display hypermethylation in the differentially methylated regions (DMRs) of Peg3 and Gnas imprinted domains, which are known to contain YY1 binding sites. Further analyses confirmed in vivo binding of Rex1 only to the unmethylated allele of these two regions. Thus, Rex1 may function as a protector for these DMRs against DNA methylation. Overall, the functional connection of Rex1 to genomic imprinting represents another case where newly made genes have co-evolved with lineage-specific phenomena.

Multiple YY1 and CTCF binding sites in imprinting control regions.

Known imprinting control regions (ICRs) contain unusual tandem arrays of DNA-binding sites for transcription factors, including YY1 for the Peg3, Gnas, and Xist/Tsix domains and CTCF for the H19/Igf2 domain. These multiple DNA-binding sites are known to be the only functionally shared and evolutionarily selected feature among these ICRs. However, it is not well understood why the imprinting control regions tend to maintain a high density of a particular transcription factor-binding site. We hypothesize that the multiplicity associated with the YY1 and CTCF binding sites may be designed for attracting and maintaining the relatively high levels of YY1 and CTCF proteins or for covering the relatively large genomic sizes of the associated ICRs. This idea remains to be tested in the near future, but it is one of the most likely explanations for all those unusual features that are associated with the functionally critical regions (ICRs) of genomic imprinting.