Tereza Ventura-Holman

Structural organization of the immunoglobulin heavy chain locus in the channel catfish: the IgH locus represents a composite of two gene clusters.

Two structurally-related genomic clusters of catfish immunoglobulin heavy chain gene segments are known. The first gene cluster contains DH and JH segments, as well as the C region exons encoding the functional Cmu. The second gene cluster contains multiple VH gene segments representing different VH families, a germline-joined VDJ, a single JH segment, and at least two pseudogene Cmu exons. It was not known whether these gene clusters were linked, nor was the organization or the location of VH segments associated within the first gene cluster known. Pulsed-field gel electrophoresis studies have been used to determine the structural organization of these gene clusters. Restriction mapping studies show that the two gene clusters are closely linked; the second gene cluster is located upstream from the first with the Cmu regions within the clusters separated by about 725kb. The clusters are in the same relative transcriptional orientation, and the results indicate that the complete IgH locus spans no more than 1000kb and may be as small as 750-800kb. VH gene segments are located both upstream and downstream of the pseudo-Cmu exons; however, no VH gene segments that hybridized with the VH specific probes were detected downstream of the functional Cmu. These studies coupled with earlier sequence analyses indicate that the catfish IgH locus arose from a massive internal duplication event. Subsequent gene rearrangement within the duplicated cluster likely resulted in the presence of the germline VDJ and the deletion of intervening V, D and J segments. Transposition by a member of the Tc1/mariner family of transposable elements appears to have led to the disruption of the duplicated Cmu.

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is up-regulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1, and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be up-regulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 over-expression in SW620 cells led to down-regulation of Fem1b protein levels. Conversely, down-regulation of RACK1 led to up-regulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein up-regulation. In conclusion, RACK1 down-regulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

Abnormal Glucose Homeostasis and Pancreatic Islet Function in Mice with Inactivation of the Fem1b Gene

ABSTRACT Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both β cells and non-β cells, and is highly expressed in INS-1E cells, a pancreatic β-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.

The Fem1c genes: conserved members of the Fem1 gene family in vertebrates.

The fem-1 gene of Caenorhabditis elegans functions in a signaling pathway that controls sex determination. Homologs of fem-1 in mammals have been characterized, consisting of two family members, Fem1a and Fem1b. We report here on Fem1c, a third member of the Fem1 gene family, in three vertebrate species: human, mouse, and zebrafish. The proteins encoded by these Fem1c genes share >99% amino acid identity between human and mouse, 79% amino acid identity between mouse and zebrafish, and end with a C-terminal Arginine residue, which distinguishes them from other FEM-1 proteins reported thus far. The human and mouse Fem1c coding regions show conservation of intron-exon structure and expression pattern in adult tissues. Human FEM1C maps to 5q22, mouse Fem1c maps to chromosome 18, and zebrafish fem1c maps to Linkage Group 8. The Fem1c genes in vertebrates may play a conserved role in the development and/or physiologic function of these organisms.

Structure and genomic organization of VH gene segments in the channel catfish: Members of different VH gene families are interspersed and closely linked

To determine the structure and organization of germline VH gene segments in the channel catfish, genomic lambda libraries were screened with cDNA probes representing different catfish VH gene families. Thirty-six VH positive genomic clones were isolated and four of these were characterized by restriction mapping and Southern blot analysis with probes specific for each known VH gene family. The four clones, representing about 65 kb of DNA, contained 21 VH segments. The average distance between segments was about 3 kb and gene segments representing different VH gene families were interspersed with each other. Dot-blot hybridization analysis of all 36 genomic clones (average insert size 16-18 kb) indicated that the average clone contained gene segments representing four different VH families. In addition, these analyses indicated that VH segments representing each VH family could be found closely linked to gene segments representing each of the other VH families. Genomic restriction fragments containing a VH segment of each gene family were sequenced. These analyses showed that the general structure of VH segments is conserved in catfish. These structural features include the presence of a leader sequence split by a short intron, an uninterrupted open reading frame encoding readily identified framework and complementarity determining regions, and a downstream recombination signal sequence represented by a consensus heptamer, a 22-24 bp spacer, and an A-rich nonamer. Upstream of the VH segments was an octamer sequence. These analyses indicate that the organization and structure of VH segments typically associated with VH loci of higher vertebrates evolved early in phylogeny at the level of the bony fishes.

CHARACTERIZATION OF A SEVENTH FAMILY OF IMMUNOGLOBULIN HEAVY CHAIN VH GENE SEGMENTS IN THE CHANNEL CATFISH, ICTALURUS PUNCTATUS

A VH gene segment that could not be assigned to any of the six known VH gene families of the channel catfish was identified in a genomic clone containing VH gene segments. This gene segment (designated VH7.1) exhibited the structural features characteristic of vertebrate VH genes, specifically potential upstream regulatory sequences, a leader sequence split by an intron, a reading frame that could be readily divided into framework and complementarity determining regions, and a 3′ recombination signal sequence. Two regions of nucleotide deletions coupled with degeneracy in the nonamer sequence indicate that this VH gene segment is a pseudogene. Genomic DN A restricted with different enzymes and hybridized under stringent conditions with probes derived from VH7.1 showed that 8‐10 bands were present in Southern blots. Reverse transcriptase PCR approaches were used to determine if any of these related sequences were expressed. Sequence analysis of cloned PCR products indicates that different VH gene segments exhibiting >80% similarity to germline VH7.1 are expressed. Multiple sequence alignments showed that the expressed cVH7a cDNA sequence shared less than 60% nucleotide similarity with representative cDNA sequences from the other known catfish VH gene families. These combined results thus fulfil the criteria for the definition of a new family of catfish VH gene segments. This newly defined, small VH family is designated VH7.

The Fem1a Gene Is Downregulated in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue neoplasm of children, and those metastatic at presentation have a poor prognosis. RMS development is related to defective skeletal muscle differentiation, involving a variety of cell signaling and transcriptional control pathways, including aberrant hedgehog signaling. Here we evaluate Fem1a, a gene highly expressed in skeletal muscle, as a candidate for involvement in RMS. Fem1a is a homolog of fem-1, which controls cell fate decisions in the sex determination pathway of Caenorhabditis elegans, a pathway with homology to mammalian hedgehog signaling. We show that Fem1a expression is activated during myocyte differentiation of C2C12 myoblasts, and this expression is largely confined to the terminally differentiating pool, not to the satellite-cell-like quiescent reserve cell pool. We find that the human homolog, FEM1A, is downregulated in all of 8 different human RMS cell lines, including those derived from embryonal and alveolar RMS. Using mouse genetic models of RMS development, we further show that Fem1a is consistently downregulated in primary RMS from Ptch1+/– mice, from p53–/– mice, from p53+/–; Ptch1+/– mice, and from HGF/SF-Ink4a/Arf–/– mice. Therefore, Fem1a downregulation may be involved in, and/or a marker of, an early cell fate defect fundamental to RMS pathogenesis.

Modulation of expression of RA-regulated genes by the oncoprotein v-erbA

Retinoic acid (RA) modulates the expression of genes involved in embryogenesis, development and differentiation processes in vertebrates. The v-erbA oncogene is known to exert a dominant-negative effect on the expression of RA-responsive genes. v-erbA belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid. While RA inhibits cell proliferation and promotes cell differentiation and apoptosis in a variety of tissues, v-erbA seems to play a role in oncogenesis, namely in the development of hepatocellular carcinoma (HCC) in a transgenic mouse model. In order to study the effect of v-erbA on RA-responsive genes, we used microarray analysis to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA for 3 h or 24 h. We have identified RA-responsive genes that are affected by v-erbA, as well as genes that are regulated by v-erbA alone. We have found that v-erbA can affect gene expression in the presence of RA and at the level of basal transcription. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC.

Fem1b antigen in the stool of ApcMin mice as a biomarker of early Wnt signaling activation in intestinal neoplasia

BACKGROUND Colorectal cancer is preventable by early detection and removal of precursor lesions. Central to early stages of colorectal neoplasia is activation of Wnt signaling, usually due to inactivation of the Apc tumor suppressor gene for which there is an established animal model, the Apc(Min) mouse. Immunodetection in stool of proteins up-regulated by aberrant Wnt signaling, within intestinal epithelial cells shed into the lumen, could be a rational approach to identify biomarkers of early intestinal neoplasia. Fem1b gene expression is up-regulated, following inactivation of Apc, in mouse intestinal epithelium. METHODS We initially screened pooled random stool samples by immunoblotting and found that we could detect, in Apc(Min) mice but not wild-type mice, a fragment of Fem1b protein with an antibody (Li-50) directed against an epitope near the middle of the protein, but not with antibodies directed against N-terminus or C-terminus epitopes. We then evaluated freshly voided individual stool samples collected on four consecutive days from four each of male and female Apc(Min) mice and their wild-type littermates. RESULTS The Fem1b antigen was detected with the Li-50 antibody in 15/16 samples from male Apc(Min) mice compared to 0/16 samples from male wild-type mice, and in 5/16 samples from female Apc(Min) mice compared to 0/16 samples from female wild-type mice. CONCLUSIONS This study provides proof-of-principle that fragments of proteins, whose expression is increased by aberrant Wnt signaling early in intestinal neoplasia, can be immunodetected in stool. Excreted Fem1b protein fragments may be a useful biomarker for epithelial Wnt signaling and early intestinal neoplasia.

High molecular weight DNA from nucleated erythrocytes for use in pulsed-field gel electrophoresis (PFGE)

Nucleated erythrocytes of lower vertebrates provide a source of genomic DNA that can be used in pulsed-field gel electrophoresis (PFGE) studies. Difficulties reported in the preparation of chicken erythrocyte DNA for analysis by PFGE suggest that the presence of hemoglobin iron may result in iron-mediated DNA degradation. We report here modifications to the procedures established for isolation of high molecular weight DNA from mammalian cells. By increasing the volume of buffers and extending the incubation periods to allow for the removal of hemoglobin iron, we have successfully prepared channel catfish erythrocyte DNA suitable for analysis by PFGE.