Andrew C. Perkins

Stem cell transcriptome profiling via massive-scale mRNA sequencing

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)+ transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF.

GLOBIN genes are regulated in a tissue-specific and developmental stage-specific manner, with the β-globin gene being the last to be activated in the β-gene cluster1. CACCC-nucleotide sequences, which bind multiple nuclear proteins, including ubiquitously expressed Spl and erythroid Krüppel-like factor (EKLF), are among the cis-regulatory sequences critical for transcription of globin and non-globin erythroid-expressed genes2-5. To determine the function of EKLF in vivo, we created mice deficient in EKLF by gene targeting6. These embryos die of anaemia during fetal liver erythropoiesis and show the molecular and haematological features of β-globin deficiency, found in β-thalassaemia. Although it is expressed at all stages, EKLF is not required for yolk sac erythropoiesis, erythroid commitment or expression of other potential target genes. Its stage-specific and β-globin-gene-specific requirement suggests that EKLF may facilitate completion of the fetal-to-adult (haemoglobin α to β) switch in humans.

ISOLATION AND CHARACTERIZATION OF THE CDNA ENCODING BKLF/TEF-2, A MAJOR CACCC-BOX-BINDING PROTEIN IN ERYTHROID CELLS AND SELECTED OTHER CELLS

CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in selected other cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF, has been shown to be required in vivo for proper expression of the adult beta-globin gene, it is dispensable for the regulation of several other globin and nonglobin erythroid cell-expressed genes. In the work described here, we searched for additional CACCC-box transcription factors that might be active in murine erythroid cells. We identified a major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, that could be distinguished from EKLF by specific antisera. Through relaxed-stringency hybridization, we obtained the cDNA encoding BKLF, a highly basic, novel zinc finger protein that is related to EKLF and other Krüppel-like members in its DNA-binding domain but unrelated elsewhere. BKLF, which is widely but not ubiquitously expressed in cell lines, is highly expressed in the midbrain region of embryonic mice and appears to correspond to the gel shift activity TEF-2, a transcriptional activator implicated in regulation of the simian virus 40 enhancer and other CACCC-box-containing regulatory elements. Because BKLF binds with high affinity and preferentially over Sp1 to many CACCC sequences of erythroid cell expressed genes, it is likely to participate in the control of many genes whose expression appears independent of the action of EKLF.

A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells

KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.

Evolution of gene function and regulatory control after whole-genome duplication: Comparative analyses in vertebrates

The significance of whole-genome duplications (WGD) for vertebrate evolution remains controversial, in part because the mechanisms by which WGD contributed to functional evolution or speciation are still incompletely characterized. Fish genomes provide an ideal context in which to examine the consequences of WGD, because the teleost lineage experienced an additional WGD soon after divergence from tetrapods and because five teleost genomes are available for comparative analysis. Here we present an integrated approach to characterize these post-duplication genomes based on genome-scale synteny, phylogenetic, temporal, and spatial gene expression and on protein sequence data. A minimum of 3%-4% of protein-coding loci have been retained in two copies in each of the five fish genomes, and many of these duplicates are key developmental genes that function as transcription factors or signaling molecules. Almost all duplicate gene pairs we examined have diverged in spatial and/or temporal expression during embryogenesis. A quarter of duplicate pairs have diverged in function via the acquisition of novel protein domains or via changes in the subcellular localization of their encoded proteins. We compared the spatial expression and protein domain architecture of zebrafish WGD-duplicates to those of their single mouse ortholog and found many examples supporting a model of neofunctionalization. WGD-duplicates have acquired novel protein domains more often than have single-copy genes. Post-WGD changes at the gene regulatory level were more common than changes at the protein level. We conclude that the most significant consequence of WGD for vertebrate evolution has been to enable more-specialized regulatory control of development via the acquisition of novel spatiotemporal expression domains. We find limited evidence that reciprocal gene loss led to reproductive isolation and speciation in this lineage.

Widespread Failure of Hematolymphoid Differentiation Caused by a Recessive Niche-Filling Allele of the Ikaros Transcription Factor

A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. Ikaros(Plastic) homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies.

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox-2.4 homeobox gene.

The murine myelomonocytic cell line WEHI‐3B exhibits ectopic expression of the genes encoding the homeobox protein, Hox‐2.4, and the myeloid growth factor, interleukin‐3 (IL‐3). We showed previously that concomitant expression of IL‐3 and Hox‐2.4 in bone marrow cells induced the development of transplantable growth factor‐independent tumours resembling the WEHI‐3B tumour. We have now investigated the effect of enforced expression of Hox‐2.4 alone. Bone marrow cells were infected with Hox‐2.4 retrovirus and then either cultured in agar or transplanted into irradiated mice. In vitro, colonies derived from virus‐infected cells readily yielded IL‐3‐dependent, non‐tumorigenic cell lines of the myelomonocytic, megakaryocytic and mast cell lineages. Surprisingly, both the establishment and maintenance of these lines required very high concentrations of IL‐3 and reduced levels promoted differentiation. Transplanted mice analysed after 3 months appeared normal but their spleen and bone marrow contained abundant provirus‐bearing progenitor cells, from which IL‐3‐dependent long‐term cell lines could readily be established in vitro. Four of 18 animals monitored for up to 12 months eventually developed clonal leukaemia, associated in three cases with IL‐3 production. Thus ectopic expression of Hox‐2.4 enhances self‐renewal of immature myeloid progenitors and progression to a fully malignant state is favoured by somatic mutations conferring autocrine production of IL‐3.

Targeted Disruption of the Basic Kruppel-Like Factor Gene (Klf3) Reveals a Role in Adipogenesis†

ABSTRACT Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.