Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression

BackgroundSelective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence.ResultsHere, we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice, assigning allele specificity to approximately two-thirds of all binding sites. While half of these are biallelic and include endogenous retrovirus (ERV) targets, the rest show monoallelic binding based either on parental origin or on genetic background of the allele. Parental-origin allele-specific binding is methylation-dependent and maps only to imprinting control differentially methylated regions (DMRs) established in the germline. We identify a novel imprinted gene, Fkbp6, which has a critical function in mouse male germ cell development. Genetic background-specific sequence differences also influence ZFP57 binding, as genetic variation that disrupts the consensus binding motif and its methylation is often associated with monoallelic expression of neighboring genes.ConclusionsThe work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.

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The online version of this article (doi:10.1186/s13059-015-0672-7) contains supplementary material, which is available to authorized users.     

Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix

Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.     

A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

Fibrillin: from domain structure to supramolecular assembly.

In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.     

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 (ICR_BS) and many other loci (non-ICR_BS). 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 ICR_BS and non-ICR_BS 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-ICR_BS, 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.     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.     

Evolution of the CDKN1C-KCNQ1 imprinted domain

Background  

Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.     

Expression patterns of the novel imprinted genes Nap1l5 and Peg13 and their non-imprinted host genes in the adult mouse brain

Recent work has implicated imprinted gene functioning in neurodevelopment and behaviour and defining the expression patterns of these genes in brain tissue has become a key prerequisite to establishing function. In this work we report on the expression patterns of two novel imprinted loci, Nap1l5 and Peg13, in adult mouse brain using in situ hybridisation methods. Nap1l5 and Peg13 are located, respectively, within the introns of the non-imprinted genes Herc3 and the Tularik1 (T1)/KIAA1882 homologue in two separate microimprinted domains on mouse chromosomes 6 and 15. These 'host' genes are highly expressed in brain and consequently we were interested in assessing their expression patterns in parallel to the imprinted genes. The brain expression of all four genes appeared to be mainly neuronal. The detailed expression profiles of Nap1l5 and Peg13 were generally similar with widespread expression that was relatively high in the septal and hypothalamic regions, the hippocampus and the cerebral cortex. In contrast, there was some degree of dissociation between the imprinted genes and their non-imprinted hosts, in that, whilst there was again widespread expression of Herc3 and the T1/KIAA1882 homologue, these genes were also particularly highly expressed in Purkinje neurons and piriform cortex. We also examined expression of the novel imprinted genes in the adrenal glands. Nap1l5 expression was localised mainly to the adrenal medulla, whilst Peg13 expression was observed more generally throughout the adrenal medulla and the outer cortical layers.     

X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.     

An internal domain of Exo70p is required for actin-independent localization and mediates assembly of specific exocyst components

The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.     

Structure of an L27 domain heterotrimer from cell polarity complex Patj/Pals1/Mals2 reveals mutually independent L27 domain assembly mode

The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.     

The Prader-Willi/Angelman imprinted domain and its control center

The present review focuses on the recent advances towards understanding the mode of operation of the imprinting center (IC) within the Prader-Willi/Angelman syndromes (PWS/AS) domain. Special emphasis is put on the elucidation of the functional interaction between the two parts of the center, AS-IC and PWS-IC. The recent studies, on which the review is based, reveal cis-acting elements and trans-acting proteins that constitute the two parts of the IC and presumably provide the molecular mechanism for this interaction. AS-IC acquires the primary imprint during gametogenesis by establishing the maternal epigenotype. The unmethylated maternal allele of the AS-IC binds, very likely, a trans-acting factor that confers methylation on the PWS-IC maternal allele after fertilization. It is assumed that the PWS-IC paternal epigenotype, once established, spreads across the entire PWS/AS domain in the soma.     

Towards unravelling the Igf2/H19 imprinted domain

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence⁽¹⁾ suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.