Non-essential function of KRAB zinc finger gene clusters in retrotransposon suppression

AbstractThe Krüppel-associated box zinc finger protein (KRAB-ZFP) family amplified and diversified in mammals by segmental duplications, but the function of the majority of this gene family remains largely unexplored due to the inaccessibility of the gene clusters to conventional gene targeting. We determined the genomic binding sites of 61 murine KRAB-ZFPs and genetically deleted in mouse embryonic stem (ES) cells five large KRAB-ZFP gene clusters encoding nearly one tenth of the more than 700 mouse KRAB-ZFPs. We demonstrate that clustered KRAB-ZFPs directly bind and silence retrotransposons and block retrotransposon-borne enhancers from gene activation in ES cells. Homozygous knockout mice generated from ES cells deleted in one of two KRAB-ZFP clusters were born at sub-mendelian frequencies in some matings, but heterozygous intercrosses could also yield knockout progeny with no overt phenotype. We further developed a retrotransposon capture-sequencing approach to assess mobility of the MMETn family of endogenous retrovirus like elements, which are transcriptionally activated in KRAB-ZFP cluster KOs, in a pedigree of KRAB-ZFP cluster KO and WT mice. We identified numerous somatic and several germ-line MMETn insertions, and found a modest increase in activity in mutant animals, but these events were detected in both wild-type and KO mice in stochastic and highly variable patterns. Our data suggests that the majority of young KRAB-ZFPs play a non-essential role in transposon silencing, likely due to the large redundancy with other KRAB-ZFPs and other transposon restriction pathways in mice.One Sentence SummaryMegabase-scale deletions of KRAB-ZFP gene clusters in mice leads to retrotransposon activation.

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Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1642 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1642-1651 ◽

Cited By ~ 240

Author(s):

M J Weiss ◽

C Yu ◽

S H Orkin

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Zinc Finger ◽

Es Cells ◽

Embryonic Stem ◽

Erythroid Cell ◽

Transactivation Domain ◽

Stage Of Development ◽

Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

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Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6755-6758.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6755-6758

Author(s):

B R Stanton ◽

S W Reid ◽

L F Parada

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Embryonic Development ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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Improved Experimental Procedures for Achieving Efficient Germ Line Transmission of Nonobese Diabetic (NOD)-Derived Embryonic Stem Cells

Experimental Diabesity Research ◽

10.1080/15438600490486877 ◽

2004 ◽

Vol 5 (3) ◽

pp. 219-226 ◽

Cited By ~ 6

Author(s):

Satoko Arai ◽

Christina Minjares ◽

Seiho Nagafuchi ◽

Toru Miyazaki

Keyword(s):

High Efficiency ◽

Germ Line ◽

Gene Knockout ◽

Es Cells ◽

Embryonic Stem ◽

Nod Mice ◽

Insulin Dependent Diabetes Mellitus ◽

Transmission Efficiency ◽

Dependent Diabetes Mellitus ◽

Specific Gene

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g.,Iddlocus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBSLett., 455, 101–104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a “pure” NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.

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Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4509-4517.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4509-4517

Author(s):

P Hasty ◽

J Rivera-Pérez ◽

C Chang ◽

A Bradley

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Mammalian Cells ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Homologous Sequence ◽

Reciprocal Recombination ◽

Replacement Vector ◽

Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

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Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽

10.1242/dev.113.3.815 ◽

1991 ◽

Vol 113 (3) ◽

pp. 815-824 ◽

Cited By ~ 6

Author(s):

M.B. Rogers ◽

B.A. Hosler ◽

L.J. Gudas

Keyword(s):

Retinoic Acid ◽

Germ Cell ◽

Cell Fate ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Preimplantation Embryos ◽

Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

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Human endogenous retrovirus protein Rec interacts with the testicular zinc-finger protein and androgen receptor

Journal of General Virology ◽

10.1099/vir.0.014241-0 ◽

2010 ◽

Vol 91 (6) ◽

pp. 1494-1502 ◽

Cited By ~ 43

Author(s):

S. Kaufmann ◽

M. Sauter ◽

M. Schmitt ◽

B. Baumert ◽

B. Best ◽

...

Keyword(s):

Androgen Receptor ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Endogenous Retrovirus ◽

Human Endogenous Retrovirus ◽

Finger Protein

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CtBP-interacting BTB Zinc Finger Protein (CIBZ) Promotes Proliferation and G1/S Transition in Embryonic Stem Cells via Nanog

Journal of Biological Chemistry ◽

10.1074/jbc.m111.333856 ◽

2012 ◽

Vol 287 (15) ◽

pp. 12417-12424 ◽

Cited By ~ 19

Author(s):

Tomonori Nishii ◽

Yu Oikawa ◽

Yasumasa Ishida ◽

Masashi Kawaichi ◽

Eishou Matsuda

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Ectopic Expression ◽

Embryonic Stem ◽

Btb Domain ◽

Constitutive Overexpression ◽

Sirna Knockdown

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

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A New Coactivator Function for Zac1's C2H2 Zinc Finger DNA-Binding Domain in Selectively Controlling PCAF Activity

Molecular and Cellular Biology ◽

10.1128/mcb.00842-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6078-6093 ◽

Cited By ~ 16

Author(s):

Anke Hoffmann ◽

Dietmar Spengler

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Zinc Finger ◽

Transcriptional Control ◽

Zinc Finger Protein ◽

Embryonic Stem ◽

Binding Domain ◽

Dna Binding Domain ◽

Histone H4 ◽

Histone H4 Acetylation

ABSTRACT The generally accepted paradigm of transcription by regulated recruitment defines sequence-specific transcription factors and coactivators as separate categories that are distinguished by their abilities to bind DNA autonomously. The C2H2 zinc finger protein Zac1, with an established role in canonical DNA binding, also acts as a coactivator. Commensurate with this function, p73, which is related to p53, is here shown to recruit Zac1, together with the coactivators p300 and PCAF, to the p21Cip1 promoter during the differentiation of embryonic stem cells into neurons. In the absence of autonomous DNA binding, Zac1's zinc fingers stabilize the association of PCAF with p300, suggesting its scaffolding function. Furthermore, Zac1 regulates the affinities of PCAF substrates as well as the catalytic activities of PCAF to induce a selective switch in favor of histone H4 acetylation and thereby the efficient transcription of p21Cip1. These results are consistent with an authentic coactivator function of Zac1's C2H2 zinc finger DNA-binding domain and suggest coactivation by sequence-specific transcription factors as a new facet of transcriptional control.

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Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽

10.1530/rep-07-0479 ◽

2008 ◽

Vol 135 (6) ◽

pp. 771-784 ◽

Cited By ~ 90

Author(s):

Fariborz Izadyar ◽

Francis Pau ◽

Joel Marh ◽

Natalia Slepko ◽

Tracy Wang ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Complex Mixture ◽

Neural Cells ◽

Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

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