POU domain genes and the immunoglobulin octamer motif in chickens

This research was undertaken to test the hypothesis that multiple POU genes exist and that POU proteins are involved in transcriptional regulation in the chicken. The POU protein Oct-2 has been hypothesized to partially regulate immunoglobulin gene expression through its interaction with the octamer motif found in the promoter of immunoglobulin genes. Transcriptional regulation of immunoglobulin genes provides a model for tissue-specific gene expression in the immune system. In searching for a chicken Oct-2 homologue, two partial chicken POU domain genes were identified. A cDNA clone encoding the POU domain of Brn-3a was isolated and showed near identity to the human Brn-3a sequence (100% at the a.a. level). Chicken Brn-3a was mapped to linkage group E48 in the East Lansing chicken genome mapping reference population. A Skn-1/Epoc-1/Oct-11 genomic clone was identified and mapped to chicken linkage group E49, to an area of synteny with human chromosome 11q23 and mouse chromosome 9. Octamer-binding protein expression patterns in multiple chicken tissues demonstrated that octamer-binding protein complexes existed in ovary, cerebrum, liver, lung, kidney, spleen, thymus, bursa, MSB1 (T cell line), and DT40 (B cell line). Every tissue had at least two octamer-binding proteins, with a total of seven unique chicken protein complexes. A comparison between mouse and chicken octamer-binding proteins identified a unique chicken octamer-binding protein, slightly faster migrating than mouse Oct-2. Chicken Oct-1 was expressed in all tissues except liver. The chicken immunoglobulin lambda light chain promoter contains an octamer motif (-106) and a TATA box (-70). Chicken B and T lymphocyte cell lines were used to determine activity of both the promoter and the octamer motif. The promoter was functional only in the B cell line and required an enhancer sequence. Mutation of the octamer motif abolished the transcriptional activity of the immunoglobulin promoter. These results suggest that the chicken immunoglobulin promoter functions similarly to its mammalian counter parts, in that the chicken immunoglobulin lambda light chain promoter plays a role in tissue-specificity and requires an enhancer and an intact octamer motif

The homeobox in vertebrate development.

http://dx.doi.org

Molecular characterization of Lhx8, an oocyte-specific transcription factor, and its interacting proteins in rainbow trout and cattle

LIM homeobox 8 (Lhx8) is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. To date, no attempts have been made to investigate the existence of any cellular factors that might interact with Lhx8 protein in oocytes and early embryos. In this study, we report the characterization of rainbow trout and bovine Lhx8 genes and identification of important germ cell-specific nuclear factors that interact with Lhx8 protein in both species. In rainbow trout, two Lhx8 genes, Lhx8a and Lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout. The open reading frame (ORF) of bovine Lhx8 gene was amplified from cDNA of a bovine fetal ovary using primers designed based on a predicted bovine Lhx8 cDNA sequence and a partial 5 \u27end transcript. The ORF of bovine Lhx8 cDNA is 1,134 bp in length encoding a protein of 377 amino acids. A splicing variant of Lhx8 (Lhx8_v1) was identified, which results from alternative splicing of exon 2 and 3, and encodes a protein of 293 amino acids. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in the splicing variant, Lhx8_v1, is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 mRNA are specifically expressed in fetal ovaries and testis but not detectable in the somatic tissues as well as in granulosa and theca cells. Lhx8 mRNA is highly abundant in GV and MII stage oocytes as well as in early stage embryos but not detectable in morula and blastocyst stage embryos. Lhx8_v1 mRNA expression is detectable in oocytes and early embryo but not in morula and blastocyst stage embryos. A GFP reporter assay revealed that Lhx8 is a nuclear protein and the predicted monopartite NLS is required for its transport into the nucleus. Direct yeast two-hybrid analysis revealed that bovine Lhx8 protein interacts with Figla, a basic helix-loop-helix transcription factor. The interaction between Lhx8 and Figla was confirmed by a co-immunoprecipitation assay. This is the first time that a direct protein-protein interaction between two germ cell-specific transcription factors essential for oocyte and follicular development is demonstrated. The study provides new information for studying the mechanisms of the regulatory roles of Lhx8 in oocyte/follicular development and early embryogenesis

Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals

Doctor of Philosophy

dissertationThe Oct1/POU2F1 transcription factor was previously thought to constitutively occupy its cognate DNA binding sites, and to regulate the expression of housekeeping genes. This stereotype led to little attention being paid to Oct1 activity in dynamic cellular responses. In 2005, Oct1-/- mouse embryonic fibroblasts were shown to be highly sensitive to oxidative and genotoxic stresses, implicating Oct1 as a stress sensor. However, the mechanism connecting stress with Oct1 transcription regulation was unknown. To identify the mechanism by which Oct1 activity is regulated by posttranslational modifications in response to stress exposure, I used affinity purification and mass spectrometry to map specific Oct1 phosphorylation, O-GlcNAc modification and ubiquitination sites. Serine 385 (Chapter 2): I identified unique mechanisms of Oct1 regulation in response to stress and during mitosis, involving two different Oct1 phosphorylation events. Following genotoxic and oxidative stress, Oct1 binding specificity is mediated by phosphorylation of S385, switching from a monomeric into a dimeric conformation on different binding sequences. I confirmed this mechanism by genome wide ChIPseq. The homologous protein Oct4, a master regulator of embryonic stem cells, uses a similar mode of regulation. Serine 335 (Chapter 3): I identified an other phosphorylation event (pS335) as a negative regulator of Oct1 binding to DNA. I found that this phosphorylation is induced iv in mitotic cells as well as in stressed ones. The phosphorylated Oct1 is enriched in the mitotic spindle poles and midbody. Phospho-Oct1 is also K11-ubiquitinated. Using several different approaches, I showed that Oct1 directly regulates mitosis after being displaced from mitotic chromatin. Threonine 255 and Serine 728 (Chapter 4): In addition to phosphorylation and ubiquitination, I also identified O-GlcNAc modification of Oct1. Using an Oct1 glycosylation defective mutant, I found that the glycosylated residues of Oct1 regulate its DNA binding and transcriptional activity. Finally, I confirmed in embryonic stem cells that Oct1 and Oct4 share binding specificity for novel multimeric as well as conventional octamer motifs (Chapter 5). Further, multimeric binding motifs recruit Oct1 and Oct4 hetero-complexes suggesting extensive crosstalk between Oct1 and Oct4 in embryonic stem cells

Murine developmental control genes.

Various strategies have been used to isolate genes that participate in the regulation of mouse development. Gene families that have been identified on the basis of their homology to motifs within Drosophila control genes or human transcription factor genes, namely homeobox (Hox), paired-box (Pax), and POU genes, can be compared with respect to gene organization, structure, and expression patterns. The functions of these genes can be analyzed molecularly in vitro and in vivo with the use of available mouse mutants or transgenic mice. In addition, it has been possible to generate gain- or loss-of-function mutations by random or targeted introduction of transgenes. Models derived from these studies can reveal the successive steps of developmental control on a genetic level