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1.  Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and their Differentiated Derivatives 
Cell Stem Cell  2012;10(5):620-634.
Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting, and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.
PMCID: PMC3348513  PMID: 22560082
2.  Notch Signaling Proteins Hes-1 and Hey-1 Bind N-box Domains in the Col2a1 Enhancer Site to Repress Chondrogenesis 
Arthritis and rheumatism  2008;58(9):2754-2763.
Notch signaling is implicated in repressing mesenchymal stem cell (MSC) chondrogenic differentiation. The mechanism of this repression and how this is modulated to permit chondrogenesis has not been elucidated.
Notch intracellular domain (NICD) protein levels were monitored via western blot throughout chondrogenic differentiation of human in MSC pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, hairy and enhancer of split 1 (Hes-1) and hairy and enhancer of split related-2 (HERP-2/Hey-1). Col2a1 and aggrecan expression was monitored via quantitative PCR. Chromatin immunoprecipitation (chIP) was utilized to test whether Hes-1 and Hey-1 bind putative N-box domains in intron 1 of Col2a1.
NICD protein levels were reduced during chondrogenesis of hMSC, which was mediated by TGFβ3. Col2a1 gene expression was repressed following overexpression of NICD (2-fold), Hes-1 (3-fold) and markedly by Hey-1 (80-fold). Hey-1 repressed aggrecan expression 10-fold, while NICD and Hes-1 had no effect. chIP studies show that endogenous Hes-1 and Hey-1 bind to two putative N-box domains adjacent to and as part of the Sox9 enhancer binding site located in intron 1 of Col2a1. The Hes-1 co-repressor protein transducin like enhancer (TLE) was displaced during chondrogenic differentiation and following TGFβ3 treatment.
These results reveal novel mechanisms by which Notch signaling represses gene expression and further define the role of TGFβ to promote chondrogenic differentiation.
PMCID: PMC2786215  PMID: 18759300
3.  Population and family studies of three disease-related polymorphic genes in systemic lupus erythematosus. 
Journal of Clinical Investigation  1995;95(4):1766-1772.
The contribution to systemic lupus erythematosus (SLE) of three lupus-associated polymorphisms (involving the C4A2 complement component, Humhv3005 and the T cell antigen receptor alpha chain gene) are investigated in 81 individuals from 14 multiplex SLE families, 41 unrelated lupus patients, and 88 unrelated healthy controls. The results show a strong association between C4A deletion and SLE in these families. While the current study confirms the previously reported association between hv3005 deletion and sporadic SLE, the study fails to support this association in familial SLE patients. Moreover, no correlation is detected between the occurrence of hv3005 deletion and C4A null alleles in lupus patients, suggesting that the effects of these genetic polymorphisms on predisposition to lupus are independent. The previously reported lupus-associated T cell receptor (TCR) alpha chain polymorphism is not detected in any of the individuals studied here. The combined data suggest that C4A null alleles predispose strongly to development of lupus, whereas the influence of hv3005 deletion is relatively weak. The results also suggest that contributions of weak susceptibility genes such as hv3005 to disease predisposition may be obscured by the effects of stronger genetic factors and thus need to be examined in patients lacking these factors.
PMCID: PMC295700  PMID: 7706484
4.  Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors. 
Journal of Clinical Investigation  1994;93(6):2545-2553.
A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.
PMCID: PMC294479  PMID: 8200991
5.  Sequence analyses of three immunoglobulin G anti-virus antibodies reveal their utilization of autoantibody-related immunoglobulin Vh genes, but not V lambda genes. 
Journal of Clinical Investigation  1992;90(6):2197-2208.
Accumulated sequence analyses of the antibody repertoire have revealed that most autoantibodies and developmentally regulated antibodies share a small set of germline Ig-variable region (V) genes. The findings have prompted speculation that certain autoantibodies are of developmental importance and may be instrumental in maintaining homeostasis of the adult antibody repertoire. In order to evaluate this hypothesis critically, it is first necessary to determine the V gene usage in human antibodies against foreign substances. Unfortunately, only a few such antibodies have had their heavy and light chains characterized. To rectify the situation, we adapted the anchored polymerase chain reaction to clone and analyze rapidly the expressed V genes for three anti-virus IgG antibodies. The results show that all three heavy chain V (Vh) genes are highly homologous to the known autoantibody-related Vh genes. In contrast, two light chain V (VL) genes of the V lambda 1 subgroup are similar to a non-autoantibody-related germline V lambda 1 gene. Taken together with the reported Vh and VL sequences of several antibodies against viruses and bacteria, the data show that many antipathogen antibodies may use the same small set of Vh genes that encode autoantibodies, but diverse VL genes that are distinct from autoantibody-related VL genes. Thus, only a small portion of the potentially functional germline Vh genes are used recurrently to generate most antibodies in a normal antibody repertoire, regardless of their reactivities with either self or non-self.
PMCID: PMC443370  PMID: 1334971
6.  Molecular basis of an autoantibody-associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases. 
Journal of Clinical Investigation  1991;88(1):193-203.
Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.
PMCID: PMC296020  PMID: 1676037

Results 1-6 (6)