Large Maf Transcription Factors: Cousins of AP-1 Proteins and Important Regulators of Cellular Differentiation

A large number of mammalian transcription factors possess the evolutionary conserved basic and leucine zipper domain (bZIP). The basic domain interacts with DNA while the leucine zipper facilitates homo- and hetero- dimerization. These factors can be grouped into at least seven families: AP-1, ATF/CREB, CNC, C/EBP, Maf, PAR, and virus-encoded bZIPs. Here, we focus on a group of four large Maf proteins: MafA, MafB, c-Maf, and NRL. They act as key regulators of terminal differentiation in many tissues such as bone, brain, kidney, lens, pancreas, and retina, as well as in blood. The DNA-binding mechanism of large Mafs involves cooperation between the basic domain and an adjacent ancillary DNA-binding domain. Many genes regulated by Mafs during cellular differentiation use functional interactions between the Pax/Maf, Sox/Maf, and Ets/Maf promoter and enhancer modules. The prime examples are crystallin genes in lens and glucagon and insulin in pancreas. Novel roles for large Mafs emerged from studying generations of MafA and MafB knockouts and analysis of combined phenotypes in double or triple null mice. In addition, studies of this group of factors in invertebrates revealed the evolutionarily conserved function of these genes in the development of multicellular organisms

Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates

<p>Abstract</p> <p>Background</p> <p>αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous <it>Cryaa </it>gene.</p> <p>Results</p> <p>Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse <it>Cryaa </it>gene. Two BAC transgenes, with EGFP insertions into the third coding exon of <it>Cryaa </it>gene, were created: the intact α<it>A-crystallin </it>148 kb BAC (αA-BAC) and αA-BAC(ΔDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the <it>Cryaa </it>gene in lens, but not outside of the lens. The number of cells expressing αA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of α<it>A-crystallin </it>locus derived from αA-BAC(ΔDCR3), 15 kb <it>Cryaa/EGFP</it>. A 15 kb region of <it>Cryaa/EGFP </it>supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of αA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing αA-crystallin in the lens pit.</p> <p>Conclusion</p> <p>We conclude that a 148 kb αA-BAC likely contains all of the regulatory regions required for αA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb <it>Cryaa/EGFP </it>region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular αA-crystallin expression. Finally, deletion of DCR3 in either αA-BAC(ΔDCR3) or <it>Cryaa </it>(15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.</p

Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues

Rybp, a polycomb complex-associated protein, is required for mouse eye development

<p>Abstract</p> <p>Background</p> <p>Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of <it>Rybp </it>in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for <it>Rybp </it>in ocular development using four <it>in vivo </it>mouse models which resulted in either the ablation or overexpression of <it>Rybp</it>.</p> <p>Results</p> <p>Our results demonstrate that loss of a single <it>Rybp </it>allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of <it>Pax6 </it>but not of <it>Pax2</it>. In addition, about one half of <it>Rybp-/- <-> Rybp+/+ </it>chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of <it>Rybp </it>in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of <it>AP-2α </it>and <it>Sox2</it>, and reduced levels of <it>βA4-crystallin </it>gene expression. Ubiquitous transgenic overexpression of <it>Rybp </it>in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development.</p> <p>Conclusion</p> <p>These studies establish <it>Rybp </it>as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as <it>BCOR</it>, <it>CBP</it>, <it>Chx10</it>, <it>Pax2</it>, <it>Pax6</it>, <it>Six3</it>, <it>Ski</it>, <it>Vax1 </it>and <it>Vax2</it>. We propose that the multiple functions for <it>Rybp </it>in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.</p

Palm is expressed in both developing and adult mouse lens and retina

BACKGROUND: Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. METHODS: The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. RESULTS: In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. CONCLUSION: Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation

Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation

These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens

Cited2 is required for the proper formation of the hyaloid vasculature and for lens morphogenesis

Cited2 is a transcriptional modulator with pivotal roles in different biological processes. Cited2-deficient mouse embryos manifested two major defects in the developing eye. An abnormal corneal-lenticular stalk was characteristic of Cited2(−/−) developing eyes, a feature reminiscent of Peters’ anomaly, which can be rescued by increased Pax6 gene dosage in Cited2(−/−) embryonic eyes. In addition, the hyaloid vascular system showed hyaloid hypercellularity consisting of aberrant vasculature, which might be correlated with increased VEGF expression in the lens. Deletion of Hif1a (which encodes HIF-1α) in Cited2(−/−) lens specifically eliminated the excessive accumulation of cellular mass and aberrant vasculature in the developing vitreous without affecting the corneal-lenticular stalk phenotype. These in vivo data demonstrate for the first time dual functions for Cited2: one upstream of, or together with, Pax6 in lens morphogenesis; and another in the normal formation of the hyaloid vasculature through its negative modulation of HIF-1 signaling. Taken together, our study provides novel mechanistic revelation for lens morphogenesis and hyaloid vasculature formation and hence might offer new insights into the etiology of Peters’ anomaly and ocular hypervascularity

Histone posttranslational modifications and cell fate determination: Lens induction requires the lysine acetyltransferases CBP and p300

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(−/−) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens

Identification of Pax6-Dependent Gene Regulatory Networks in the Mouse Lens

Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99–119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed