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Biomedical subjects

J R Friedman

Publications and source records attributed to J R Friedman.

At least 19 recordsLinked to original sources
PubMed2006

The Foxa family of transcription factors in development and metabolism.

The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors has been the subject of genetic and biochemical study for over 15 years. During this time its three members, Foxa1, Foxa2 and Foxa3, have been found to play important roles in multiple stages of mammalian life, beginning with early development, continuing during organogenesis, and finally in metabolism and homeostasis in the adult. Foxa2 is required for the formation of the node and notochord, and in its absence severe defects in gastrulation, neural tube patterning, and gut morphogenesis result in embryonic lethality. Foxa1 and Foxa2 cooperate to establish competence in foregut endoderm and are required for normal development of endoderm-derived organs such as the liver, pancreas, lungs, and prostate. In post-natal life, members of the Foxa family control glucose metabolism through the regulation of multiple target genes in the liver, pancreas, and adipose tissue. Insight into the unique molecular basis of Foxa function has been obtained from recent genetic and genomic data, which identify the Foxa proteins as 'pioneer factors' whose binding to promoters and enhancers enable chromatin access for other tissue-specific transcription factors.

Animals↗
PubMed2001

Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD.

Macromolecular complexes containing histone deacetylase and ATPase activities regulate chromatin dynamics and are vitally responsible for transcriptional gene silencing in eukaryotes. The mechanisms that target these assemblies to specific loci are not as well understood. We show that the corepressor KAP-1, via its PHD (plant homeodomain) and bromodomain, links the superfamily of Krüppel associated box (KRAB) zinc finger proteins (ZFP) to the NuRD complex. We demonstrate that the tandem PHD finger and bromodomain of KAP-1, an arrangement often found in cofactor proteins but functionally ill-defined, form a cooperative unit that is required for transcriptional repression. Substitution of highly related PHD fingers or bromodomains failed to restore repression activity, suggesting high specificity in their cooperative function. Moreover, single amino acid substitutions in either the bromodomain or PHD finger, including ones that mimic disease-causing mutations in the hATRX PHD finger, abolish repression. A search for effectors of this repression function yielded a novel isoform of the Mi-2alpha protein, an integral component of the NuRD complex. Endogenous KAP-1 is associated with Mi-2alpha and other components of NuRD, and KAP-1-mediated silencing requires association with NuRD and HDAC activity. These data suggest the KRAB-ZFP superfamily of repressors functions to target the histone deacetylase and chromatin remodeling activities of the NuRD complex to specific gene promoters in vivo.

Amino Acid Sequence↗
PubMed2000

Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain.

The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K(d) of 142 nm. The fitted R(max) indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.

Amino Acid Sequence↗
PubMed2000

Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions.

The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene silencing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface. Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity. We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components. Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain. A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain. The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions. KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity. Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding. Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain. The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition. These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins.

Amino Acid Motifs↗
PubMed2000

An engineered PAX3-KRAB transcriptional repressor inhibits the malignant phenotype of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene.

The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is dependent on the FKHR activation domain. We have fused the KRAB repression module to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.

Amino Acid Motifs↗
PubMed1999

KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing.

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

3T3 Cells↗
PubMed1996

KAP-1, a novel corepressor for the highly conserved KRAB repression domain.

The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING-B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors.

Amino Acid Sequence↗
PubMed1994

Krüppel-associated boxes are potent transcriptional repression domains.

The Krüppel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic alpha-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krüppel-class Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown. Here it is shown that the KRAB domain functions as a DNA binding-dependent transcriptional repressor when fused to a heterologous DNA-binding domain from the yeast GAL4 protein. A 45-aa segment containing one of the predicted KRAB amphipathic helices was necessary and sufficient for repression. Amino acid substitutions in the predicted helix abolished the repression function. These results assign a function, transcriptional repression, to the highly conserved KRAB box and define a minimal repression domain which may aid in identifying mechanisms of repression.

3T3 Cells↗
PubMed1993

Five techniques for single tooth replacement on endosseous root-form implants.

Internally hexed and threaded root-form implants provide the clinician with many new treatment options for single tooth restorations. Coupled with an interlocking hex on the abutment, they provide stability for single tooth restorations while maintaining the options of screw or cement retention. Five techniques for fabricating single tooth restorations are presented. Clinical and laboratory evaluations of implant angulation, quantity and quality of the gingival tissues, and limitations in vertical height are discussed in relation to design, abutment selection, and laboratory procedures for each restoration. These techniques permit design of each restoration to meet the unique anatomic, functional and esthetic needs. Both screw-retained and cement-retained abutments and prostheses are discussed.

Dental Abutments↗
PubMed1993

Single tooth replacement with a combined post-and-crown implant prosthesis: a case report.

A patient desiring restoration of an individual second molar requested an osseointegrated implant. However, this patient lacked sufficient vertical space to accommodate a cemented crown, and did not want to compromise the adjacent dentition. A combination post-and-crown prosthesis effectively restored function and clinically acceptable aesthetics for this patient. The techniques for fabrication and rationale for this type of prosthesis are discussed.

Adult↗
PubMed1989

Correlation of estimated renal function parameters versus 24-hour creatinine clearance in ambulatory elderly.

This study evaluates the correlation between estimated renal function parameters (ie, creatinine clearance by the Cockcroft-Gault and Lott-Hayton formulas, serum creatinine and blood urea nitrogen) and 24-hour creatinine clearance in 15 young and 15 elderly subjects. Correlation coefficients (r) for the elderly group comparing Cockcroft-Gault and Lott-Hayton against 24-hour creatinine clearance were 0.73 (P less than .005) and 0.60 (P less than .02) respectively, and r for the young subjects were 0.37 (P greater than .05) and 0.57 (P less than .05), respectively. In 13 elderly subjects with creatinine clearance of 60 mL/minute or less, four (31%) had blood urea nitrogen of 20 mg/dL or less and 13 (100%) had serum creatinine between 1.0-1.5 mg/dL. We conclude that the formulas of Cockcroft-Gault and Lott-Hayton are valid in ambulatory elderly patients who are functionally independent without severe underlying disease, have normal body weight and are not on medication affecting renal function. Furthermore, in this select elderly population, an apparently normal serum creatinine (1.0-1.5 mg/dL) and blood urea nitrogen (20 mg/dL or less) may frequently represent a 24-hour creatinine clearance of 60 mL/minute or less.

Adult↗
PubMed1989

Osseointegrated implants: five systems. The Core-Vent implant system.

The developmental concepts of the Core-Vent System have been shaped by a desire to provide practicing dentists with a user-friendly system of oral implantology and restorative procedures. By providing implants in varying diametres, lengths and designs with a simplified surgical protocol, a true system of fitting the implant to the patient, not the patient to the implant, has evolved to meet the clinical needs of both dentists and patient. Standardized prosthetics enable similar restorations to be performed on all implants in the system. Bendable titanium inserts and castable abutments allow the optimum achievement of esthetics, regardless of the angle of implant placement.

Dental Abutments↗
PubMed1985

Stereoacuity in patients with optic nerve disease.

Twenty-eight patients were given the Titmus stereoacuity test (TST). Seventeen patients had documented optic nerve disease, and 11 had no known optic nerve disease. Thirteen of 17 patients (76%) with known optic nerve disease failed to achieve their expected TST scores, while only three of 11 (27%) of the patients without optic nerve disease fell into this category. Patients with reduced visual acuity secondary to optic nerve disease showed a disproportionately greater reduction in their TST scores than would be expected, based on Snellen's visual acuities.

Adult↗