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Biomedical subjects

W C Forrester

Publications and source records attributed to W C Forrester.

At least 19 recordsLinked to original sources
PubMed2002

The Ror receptor tyrosine kinase family.

Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction, and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes.

Amino Acid Sequence↗
PubMed2001

The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation.

The incidence of osteosarcoma is increased 500-fold in patients who inherit mutations in the RB gene. To understand why the retinoblastoma protein (pRb) is specifically targeted in osteosarcoma, we studied its function in osteogenesis. Loss of pRb but not p107 or p130 blocks late osteoblast differentiation. pRb physically interacts with the osteoblast transcription factor, CBFA1, and associates with osteoblast-specific promoters in vivo in a CBFA1-dependent fashion. Association of pRb with CBFA1 and promoter sequences results in synergistic transactivation of an osteoblast-specific reporter. This transactivation function is lost in tumor-derived pRb mutants, underscoring a potential role in tumor suppression. Thus, pRb functions as a direct transcriptional coactivator promoting osteoblast differentiation, which may contribute to the targeting of pRb in osteosarcoma.

Animals↗
PubMed1999

Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer-promoter interactions.

The immunoglobulin intragenic mu enhancer region acts as a locus control region that mediates transcriptional activation over large distances in germ line transformation assays. In transgenic mice, but not in transfected tissue culture cells, the activation of a variable region (V(H)) promoter by the mu enhancer is dependent on flanking nuclear matrix attachment regions (MARs). Here, we examine the effects of DNA methylation, which occurs in early mouse development, on the function of the mu enhancer and the MARs. We find that methylation of rearranged mu genes in vitro, before transfection, represses the ability of the mu enhancer to activate the V(H) promoter over the distance of 1.2 kb. However, methylation does not affect enhancer-mediated promoter activation over a distance of 150 bp. In methylated DNA templates, the mu enhancer alone induces only local chromatin remodeling, whereas in combination with MARs, the mu enhancer generates an extended domain of histone acetylation. These observations provide evidence that DNA methylation impairs the distance independence of enhancer function and thereby imposes a requirement for additional regulatory elements, such as MARs, which facilitate long-range chromatin remodeling.

Acetylation↗
PubMed1999

A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division.

Ror kinases are a family of orphan receptors with tyrosine kinase activity that are related to muscle specific kinase (MuSK), a receptor tyrosine kinase that assembles acetylcholine receptors at the neuromuscular junction. Although the functions of Ror kinases are unknown, similarities between Ror and MuSK kinases have led to speculation that Ror kinases regulate synaptic development. Here we show that the Caenorhabditis elegans gene cam-1 encodes a member of the Ror kinase family that guides migrating cells and orients the polarity of asymmetric cell divisions and axon outgrowth. We find that tyrosine kinase activity is required for some of the functions of CAM-1, but not for its role in cell migration. CAM-1 is expressed in cells that require its function, and acts cell autonomously in migrating neurons. Overexpression and loss of cam-1 function result in reciprocal cell-migration phenotypes, indicating that levels of CAM-1 influence the final positions of migrating cells. Our results raise the possibility that Ror kinases regulate cell motility and asymmetric cell division in organisms as diverse as nematodes and mammals.

Amino Acid Sequence↗
PubMed1998

Identification of Caenorhabditis elegans genes required for neuronal differentiation and migration.

To understand the mechanisms that guide migrating cells, we have been studying the embryonic migrations of the C. elegans canal-associated neurons (CANs). Here, we describe two screens used to identify genes involved in CAN migration. First, we screened for mutants that died as clear larvae (Clr) or had withered tails (Wit), phenotypes displayed by animals lacking normal CAN function. Second, we screened directly for mutants with missing or misplaced CANs. We isolated and characterized 30 mutants that defined 14 genes necessary for CAN migration. We found that one of the genes, ceh-10, specifies CAN fate. ceh-10 had been defined molecularly as encoding a homeodomain protein expressed in the CANs. Mutations that reduce ceh-10 function result in Wit animals with CANs that are partially defective in their migrations. Mutations that eliminate ceh-10 function result in Clr animals with CANs that fail to migrate or express CEH-23, a CAN differentiation marker. Null mutants also fail to express CEH-10, suggesting that CEH-10 regulates its own expression. Finally, we found that ceh-10 is necessary for the differentiation of AIY and RMED, two additional cells that express CEH-10.

Animals↗
PubMed1997

Extension of chromatin accessibility by nuclear matrix attachment regions.

Transcription of the variable region of the rearranged immunoglobulin mu gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer. As this mu gene enhancer can establish local areas of accessible chromatin, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking mu enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The mu enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibility correlates with extended demethylation of the gene construct but not with whether it is being actively transcribed. MARs thus collaborate with the mu enhancer to generate an extended domain of accessible chromatin.

Animals↗
PubMed1997

Genes necessary for C. elegans cell and growth cone migrations.

The migrations of cells and growth cones contribute to form and pattern during metazoan development. To study the mechanisms that regulate cell motility, we have screened for C. elegans mutants defective in the posteriorly directed migrations of the canal-associated neurons (CANs). Here we describe 14 genes necessary for CAN cell migration. Our characterization of the mutants has led to three conclusions. First, the mutations define three gene classes: genes necessary for cell fate specification, genes necessary for multiple cell migrations and a single gene necessary for final positioning of migrating cells. Second, cell interactions between the CAN and HSN, a neuron that migrates anteriorly to a position adjacent to the CAN, control the final destination of the HSN cell body. Third, C. elegans larval development requires the CANs. In the absence of CAN function, larvae arrest development, with excess fluid accumulating in their pseudocoeloms. This phenotype may reflect a role of the CANs in osmoregulation.

Animals↗
PubMed1996

The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell migration.

The assembly of the nervous system in the nematode C. elegans requires the directed migrations of cells and growth comes along the anteroposterior and dorsoventral body axis. We show here that the gene vab-8 is essential for most posteriorly directed migrations of cells and growth cones. Mutations in vab-8 disrupt fourteen of seventeen posteriorly directed migrations, but only two of seventeen anteriorly directed and dorsoventral migrations. For two types of neurons that extend axons both anteriorly and posteriorly, vab-8 mutations disrupt only the growth of the posteriorly directed axon. vab-8 encodes two genetic activities that function in the guidance of different migrations. Our results suggest that most posteriorly directed cell and growth cone migrations are guided by a common mechanism involving the vab-8 gene.

Alleles↗
PubMed1995

Inhibition of an erythroid differentiation switch by the helix-loop-helix protein Id1.

The Id proteins function as negative regulators of basic-helix-loop-helix transcription factors, which play important roles in determination of cell lineage and in tissue-specific differentiation. Down-regulation of Id1 mRNA is associated with dimethyl sulfoxide-induced terminal differentiation of mouse erythroleukemia cells. To examine the significance of Id1 down-regulation in erythroid differentiation, we generated stable mouse erythroleukemia cell lines that constitutively express a "marked" form of the murine Id1 gene. Terminal erythroid differentiation was inhibited in these lines, as indicated by a block in activation of the erythroid-specific genes alpha-globin, beta-globin, and band 3 and continued proliferation in the presence of dimethyl sulfoxide. Interestingly, this block occurred even in the presence of normal levels of the lineage-specific transcription factors GATA-1, NF-E2, and EKLF. Constitutive expression of Id1 did not interfere with DNase I hypersensitivity at site HS2 of the locus control region, expression of the erythropoietin receptor gene, or down-regulation of the endogenous Id1 or c-myc genes. The differentiation block is reversible in these lines and can be rescued by fusion with human erythroleukemia cells. These findings suggest that in vivo, Id1 functions as an antagonist of terminal erythroid differentiation.

Amino Acid Sequence↗
PubMed1994

Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions.

Transcription of the immunoglobulin mu heavy chain locus is regulated by an intronic enhancer that is flanked on both sides by nuclear matrix attachment regions (MARs). These MARs have now been shown to be essential for transcription of a rearranged mu gene in transgenic B lymphocytes, but they were not required in stably transfected tissue culture cells. Normal rates of transcriptional initiation at a variable region promoter and the formation of an extended deoxyribonuclease I (DNase I)--sensitive chromatin domain were dependent on MARs, although DNase I hypersensitivity at the enhancer was detected in the absence of MARs. Thus, transcriptional activation of the mu gene during normal lymphoid development requires a synergistic collaboration between the enhancer and flanking MARs.

Animals↗
PubMed1993

The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation.

Factor access in chromatin has been proposed to be facilitated by transcriptional enhancers. With the aim of uncoupling factor access from transcriptional stimulation by protein-protein contacts, we analyzed the potential of enhancer fragments to confer accessibility upon a linked promoter for prokaryotic T7 RNA polymerase. Access to the T7 promoter in pre-B cells from transgenic mice was examined by transcribing chromatin of isolated nuclei with T7 RNA polymerase. A 95-bp immunoglobulin mu enhancer core element was necessary and sufficient to confer accessibility upon the T7 promoter independent of its chromosomal position. This enhancer-dependent factor access could be uncoupled from an active transcriptional state of the transgene and was not accompanied by the formation of pronounced DNase I hypersensitive sites. Additional mu enhancer sequences comprising previously identified matrix attachment regions and a cryptic promoter were required to induce DNase I hypersensitivity. Together, these data provide evidence that the 95-bp mu enhancer core can establish localized factor access in nuclear chromatin independent of detectable transcription by endogenous polymerases and suggest that multiple steps are involved in the alteration of chromatin structure.

Animals↗
PubMed1992

Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex regulatory element that controls the erythroid-specific expression of all cis-linked globin genes. The LCR is composed of five DNase I hypersensitive sites (HS) spanning 16 kb and located greater than 50 kb upstream of the beta-globin gene on chromosome 11. Constructs containing all or some of these HS have been shown to produce high-level erythroid-specific expression of linked genes in transgenic mice and transfected cells. In all transgenic and transfection experiments reported to date, however, the spatial relationships between the LCR and globin genes have been disrupted. We have used homologous recombination (HR) as an approach to gain insights into the potential interactions between the LCR and globin genes in their native locations. A hygromycin B resistance (hygro(R)) gene was inserted into the human beta-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human beta-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the beta-globin gene is transcriptionally inactive and not inducible. In contrast, the hygro(R) gene within the LCR is inducible, whereas randomly integrated hygro(R) genes are not inducible in these cells. The chromatin structure of the targeted locus is also altered. A new DNase I HS is present in the enhancer/promoter of the hygro(R) gene inserted into the LCR, whereas a HS normally present in the LCR 3' to the insertion is lost and the beta-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.

Base Sequence↗
PubMed1990

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

Chromatin↗
PubMed1990

Translocation of an erythroid-specific hypersensitive site in deletion-type hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) can involve large deletions which eliminate the 3' end of the beta-like globin gene cluster and more than 70 kilobases (kb) of flanking DNA. Blot hybridization revealed a DNase I-hypersensitive site extending from 1.1 to 1.4 kb downstream of the HPFH-1 3' deletion endpoint. The site was found in normal fetal and adult nucleated erythroid cells and in two erythroleukemia cell lines but not in nonerythroid cells and tissues. Simian virus 40 core enhancer-like sequences were found nonrandomly distributed within the boundaries of the site, which is contained in a fragment of known enhancer activity (E. A. Feingold and B. G. Forget, Blood, in press). A second hypersensitive site was found 0.5 kb upstream of the HPFH-1 3' deletion endpoint but was not erythroid specific. A third site, most prominent in fetal liver-derived erythroid cells, was found 1 kb upstream of the HPFH-2 deletion endpoint. As predicted by the locations of the deletion endpoints, the first two sites were translocated to within 12 kb of the A gamma gene in erythroid colonies derived from an HPFH-2 heterozygote and in hybrid mouse-human erythroid cells carrying the HPFH-2 deletion chromosome. Further analysis of this region showed that it was DNase I sensitive in erythroid and myeloid cells, indicating that it resides in an open chromatin domain. These observations suggest that alterations of chromatin structure flanking the fetal globin genes may contribute to abnormal gene regulation in deletion-type HPFH.

Adult↗
PubMed1989

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.

Animals↗
PubMed1989

The human beta-globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice.

We linked a 3.3-kilobase fragment containing the entire A gamma-globin gene together with 1.3 kilobases of 5' flanking and 0.37 kilobase of 3' flanking DNA to a 2.5-kilobase fragment containing four of the developmentally stable hypersensitive sites normally located in the 5' region of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and its expression was analyzed in the primitive and definitive erythroid cells, as well as the brain of 14-day embryos. All six transgenic individuals that contained intact copies of the construct expressed the transgene in an erythroid-specific fashion. Expression was observed in both primitive and definitive erythroid cells. This is in marked contrast to previous transgenic mice experiments using the same A gamma-globin gene fragment in isolation, where expression was restricted to primitive erythroid cells. Our results show that the region containing the developmentally stable globin locus hypersensitive sites changes the developmental stage specificity of a human fetal globin gene in transgenic mice. These observations imply that sequences additional to those used here are involved in the developmental control of fetal globin gene expression in vivo. The ability to express fetal globin in adult erythroid cells allows one to consider using fetal globin genes for gene therapy of sickle cell disease.

Animals↗
PubMed1988

Asynchronous DNA replication within the human beta-globin gene locus.

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.

Cell Cycle↗