Olga Ermakova

C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation

The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPα and C/EBPβ are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPα and β in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and ΔNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for ΔNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPα/β induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic–erythroid and granulocytic–monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic–erythroid versus granulocytic–monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Distinct C/EBPα motifs regulate lipogenic and gluconeogenic gene expression in vivo

The C/EBPα transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock‐in mutagenesis to identify C/EBPα domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline–histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl‐CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP‐1 as well as C/EBPα, and the PHR was required for C/EBPα‐SREBP transcriptional synergy. In contrast, the highly conserved C/EBPα CR4 domain was found to undergo liver‐specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC‐1α, leading to glucose intolerance. Our results show that pathway‐specific metabolic regulation can be achieved through a single transcription factor containing context‐sensitive regulatory domains, and indicate C/EBPα phosphorylation as a PGC‐1α‐independent mechanism for regulating hepatic gluconeogenesis.

Role of the EBNA-1 Protein in Pausing of Replication Forks in the Epstein-Barr Virus Genome

We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.

Sensitized phenotypic screening identifies gene dosage sensitive region on chromosome 11 that predisposes to disease in mice

The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease‐relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase‐sized chromosomal deletion and deficiency lines carrying altered copy numbers of ∼30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high‐fat diet‐induced metabolic syndrome, antigen‐induced immune hypersensitivity, ApoE‐induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen‐induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes.

Changes in Replication, Nuclear Location, and Expression of the Igh Locus after Fusion of a Pre-B Cell Line with a T Cell Line

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3′ end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3′ to 5′ unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) × BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3′ Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.

Insights into specificity, redundancy and new cellular functions of C/EBPa and C/EBPb transcription factors through interactome network analysis.

BACKGROUND C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each others absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies. METHODS To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification (TAP) and Mass Spectrometry (MS). To unravel the functional features of C/EBPa and C/EBPb proteomes we studied the statistical enrichment of binding partners related to Gene Ontology (GO) terms and KEGG pathways. RESULTS Our data confirmed that the C/EBPa and C/EBPb regulate biological processes like cell proliferation, apoptosis and transformation. We found that both C/EBPa and C/EBPb are involved in other cellular pathways such as RNA maturation, RNA splicing and DNA repair. Specific interactions of C/EBPa with MRE11, RUVBL1 and RUVBL2 components of DNA repair system were confirmed by co-immunoprecipitation assays. CONCLUSIONS Our comparative analysis of the C/EBPa and C/EBPb proteomes provides an insight for understanding both their redundant and specific roles in cells indicating their involvement in new pathways. Such novel predicted functions are relevant to normal cellular processes and disease phenotypes controlled by these transcription factors. GENERAL SIGNIFICANCE Functional characterization of C/EBPa and C/EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.

Wnt/β-Catenin Signaling Induces Integrin α4β1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4+ T cells in the CNS are not known. In this article, we report that abnormal β-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced β-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4β1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of β-catenin in mature naive T cells was sufficient to drive integrin α4β1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4β1 reduced the abnormal T cell presence in the CNS of β-catenin–expressing mice. Together, these results implicate deregulation of the Wnt/β-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.

Construction and phenotypic analysis of mice carrying a duplication of the major histocompatibility class I (MHC-I) locus.

Copy number variation (CNV) has been associated increasingly with altered susceptibility to human disease. Large CNVs are likely to incur disease risk or resilience via predictable changes in gene dosage that are relatively straightforward to model using chromosomal engineering in mice. The classical class I major histocompatibility locus (MHC-I) contains a dense set of genes essential for innate immune system function in vertebrates. MHC-I genes are highly polymorphic and genetic variation in the region is associated with altered susceptibility to a wide variety of common diseases. Here we investigated the role of gene dosage within MHC-I on susceptibility to disease by engineering a mouse line carrying a 1.9-Mb duplication of this region [called Dp(MHC-I)]. Extensive phenotypic analysis of heterozygous (3N) Dp(MHC-I) animals did not reveal altered blood and stem cell parameters, susceptibility to high-fat diet, death by cancer, or contact dermatitis. However, several measures of disease severity in a model of atherosclerosis were improved, suggesting dosage-sensitive modulators of cardiovascular disease. Homozygous Dp(MHC-I)/Dp(MHC-I) mice demonstrated embryonic lethality. These mice serve as a model for studying the consequences of targeted gene dosage alteration in MHC-I with functional and evolutionary implications.