The specification and maintenance of renal cell types by epigenetic factors

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the roles of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish and humans. Although Pax2 and the related gene, Pax8, are essential for early intermediate mesoderm specification and are found in the epithelial lineage arising from that mesoderm, how these proteins regulate cell lineage restriction and gene expression patterns has remained obscure. Our recent data, suggests that Pax proteins help establish chromatin domains within cell lineages by providing the locus and tissue specificity for epigenetic imprinting complexes that modify histones. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. Given the prevalence of Pax2 expression in kidney development and in kidney disease, we now need to address the effects of epigenetics on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.Key words: kidney development, Pax2, PTIP, histone methyltransferase     

Proneural genes and the specification of neural cell types

Certain morphological, physiological and molecular characteristics are shared by all neurons. However, despite these similarities, neurons constitute the most diverse cell population of any organism. Recently, considerable attention has been focused on identifying the molecular mechanisms that underlie this cellular diversity. Parallel studies in Drosophila and vertebrates have revealed that proneural genes are key regulators of neurogenesis, coordinating the acquisition of a generic neuronal fate and of specific subtype identities that are appropriate for the location and time of neuronal generation. These studies reveal that, in spite of differences between invertebrate and vertebrate neural lineages, Drosophila and vertebrate proneural genes have remarkably similar roles.     

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.     

Genomic specification and epigenetic regulation of eukaryotic DNA replication origins

Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.     

The role of DMDs in the maintenance of epigenetic states

An important aspect of genome reprogramming is the establishment and maintenance of gamete-specific DNA methylation patterns that distinguish the parental alleles of imprinted genes. Disrupting the accurate transmission of genomic imprints by interfering with these methylation patterns causes severe defects in fetal growth and development. The inheritance of sex-specific DNA methylation patterns from both parents is thus a fundamental molecular definition of genomic imprinting. The other cardinal aspect is the regulation of imprinted gene expression over a long genomic distance, spanning a few clustered imprinted genes. There is converging experimental evidence that differentially methylated domains (DMDs), located in non-coding regions of imprinted genes, are involved in both processes. As such, DMDs are the imprinting backbone upon which the fundamental processes of sex-specific methylation and imprinted gene expression are built.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Prognostic factors in renal cell carcinoma

We studied 569 cases of renal cell carcinoma in the files of the Department of Pathology of the Norwegian Radium Hospital from 1964 to 1974. A nephrectomy had been performed in all cases. Clinical information on sex, age, survival time and metastases was traced. The histological slides were examined and tumour growth pattern, cell type, cell shape, nuclear atypia, abnormal nucleoli, nuclear grade, vascular invasion and tumour demarcation were all evaluated. Besides well-known prognostic factors such as tumour stage, presence or absence of metastases and vascular invasion, nuclear grade was found to be a useful prognostic factor. Younger patients were found to do better than older, and women better than men. Smaller tumours carried a better prognosis than larger and clear cell tumours had a better prognosis than those composed of eosinophilic or basophilic cells. The presence of spindle cells was a bad prognostic omen.     

Theoretical analysis of epigenetic cell memory by nucleosome modification

Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.     

The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance

BACKGROUND: The C-terminal tails of spliceosomal Sm proteins contain symmetrical dimethylarginine (sDMA) residues in vivo. The precise function of this posttranslational modification in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and pre-mRNA splicing remains largely uncharacterized. Here, we examine the organismal and cellular consequences of loss of symmetric dimethylation of Sm proteins in Drosophila. RESULTS: Genetic disruption of dart5, the fly ortholog of human PRMT5, results in the complete loss of sDMA residues on spliceosomal Sm proteins. Similarly, valois, a previously characterized grandchildless gene, is also required for sDMA modification of Sm proteins. In the absence of dart5, snRNP biogenesis is surprisingly unaffected, and homozygous mutant animals are completely viable. Instead, Dart5 protein is required for maturation of spermatocytes in males and for germ-cell specification in females. Embryos laid by dart5 mutants fail to form pole cells, and Tudor localization is disrupted in stage 10 oocytes. Transgenic expression of Dart5 exclusively within the female germline rescues pole-cell formation, whereas ubiquitous expression rescues sDMA modification of Sm proteins and male sterility. CONCLUSIONS: We have shown that Dart5-mediated methylation of Sm proteins is not essential for snRNP biogenesis. The results uncover a novel role for dart5 in specification of the germline and in spermatocyte maturation. Because disruption of both dart5 and valois causes the specific loss of sDMA-modified Sm proteins and studies in C. elegans show that Sm proteins are required for germ-granule localization, we propose that Sm protein methylation is a pivotal event in germ-cell development.     

Evolvability of cell specification mechanisms

The architecture of gene action during development is relevant to phenotypic evolution as it links genotype to morphological phenotype. Analysis of development at the level of cell fate specification mechanisms illuminates some of the properties of developmental evolution. In this article, we first review examples of evolutionary change in mechanisms of cell fate specification, with an emphasis on evolution in the dependence on inductive signaling and on evolution of the mechanisms that result in spatial asymmetries. We then focus on properties of development that bias possible phenotypic change and present how the distribution of phenotypes that are available by mutational change of the starting genotype can be experimentally tested by systematic mutagenesis. We finally discuss ways in which selection pressures on phenotypes can be inferred from a comparison of the phenotypic spectrum found on mutation with that found in the wild.     

Male germ cell specification and differentiation

Understanding the mechanisms by which the germline is induced and maintained should lead to a broader understanding of the means by which pluripotency is acquired and maintained. In this review, two major aspects of male germ cell development are discussed: underlying mechanisms for induction and maintenance of primordial germ cells and the basic signaling pathways that determine spermatogonial cell fate.