ORCID Profile
0000-0002-0311-6725
Current Organisations
i-Synapse PTY LTD
,
Fred Hutchinson Cancer Research Center
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Publisher: Elsevier BV
Date: 12-2014
Publisher: Springer Science and Business Media LLC
Date: 13-11-2012
Abstract: We have recently described a method for the construction of an informative gene expression correlation landscape for a single tissue, longissimus muscle (LM) of cattle, using a small number (less than a hundred) of erse s les. Does this approach facilitate interspecies comparison of networks? Using gene expression datasets from LM s les from a single postnatal time point for high and low muscling sheep, and from a developmental time course (prenatal to postnatal) for normal sheep and sheep exhibiting the Callipyge muscling phenotype gene expression correlations were calculated across subsets of the data comparable to the bovine analysis. An “Always Correlated” gene expression landscape was constructed by integrating the correlations from the subsets of data and was compared to the equivalent landscape for bovine LM muscle. Whilst at the high level apparently equivalent modules were identified in the two species, at the detailed level overlap between genes in the equivalent modules was limited and generally not significant. Indeed, only 395 genes and 18 edges were in common between the two landscapes. Since it is unlikely that the equivalent muscles of two closely related species are as different as this analysis suggests, within tissue gene expression correlations appear to be very sensitive to the s les chosen for their construction, compounded by the different platforms used. Thus users need to be very cautious in interpretation of the differences. In future experiments, attention will be required to ensure equivalent experimental designs and use cross-species gene expression platform to enable the identification of true differences between different species.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 06-03-2020
Abstract: We provide an empirical model to evaluate and rank data obtained from preclinical tuberculosis vaccine studies.
Publisher: Frontiers Media SA
Date: 17-03-2023
DOI: 10.3389/FIMMU.2023.1146997
Abstract: Hepatitis-associated aplastic anemia (HAAA) is a rare variant of acquired aplastic anemia characterized with a syndrome of bone marrow failure after hepatitis. We retrospectively analyzed the outcomes of consecutive severe HAAA patients who received immunosuppressive therapy (IST, n = 70), matched-sibling donor hematopoietic stem cell transplantation (MSD-HSCT, n = 26) or haploidentical-donor (HID) HSCT ( n = 11) as the first-line treatment. In the IST group, the hematologic response (HR) rate was 55.71% at 6 months. In contrast, HSCT recipients exhibited significantly more rapid and sustained hematopoiesis (HR 76.92%, 96.15% and 96.15% at 3, 6 and 12months, respectively). The 5-year overall survival (OS) was not different among IST (83.7 ± 4.9%), MSD-HSCT (93.3 ± 6.4%) and HID-HSCT group (80.8 ± 12.3%). Compared with IST, MSD and HID-HSCT demonstrated a trend of superiority in the estimated 5-year failure-free survival rates (93.3 ± 6.4% vs 64.3 ± 6.0%, p = 0.05 80.8 ± 12.3% vs 64.3 ± 6.0%, p = 0.57). In subsequent stratified analysis on age, we found that HID-HSCT showed its efficacy and safety among young patients. In sum, MSD-HSCT remains first-line treatment choice for HAAA, whereas HID-HSCT represents an alternative treatment choice in addition to IST for young patients (& 40 years) without a matched sibling donor.
Publisher: Public Library of Science (PLoS)
Date: 13-10-2020
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550138.V1
Abstract: Abstract The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8 sup + /sup T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8 sup + /sup T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation. /
Publisher: Springer Science and Business Media LLC
Date: 24-08-2020
Publisher: American Society of Hematology
Date: 05-12-2019
Abstract: DCs are the principal source of IL-6 dysregulation after alloSCT. IL-6–dependent GVHD is driven by classical signaling of IL-6R on donor T cells but is regulated by trans signaling.
Publisher: Research Square Platform LLC
Date: 06-10-2022
DOI: 10.21203/RS.3.RS-2064212/V1
Abstract: Background: It has been well-documented that haplo-identical hematopoietic stem cell transplantation (HID-HSCT) can provide outcomes comparable to conventional matched sibling donor (MSD) HSCT, however, little is known about the effects on quality of life (QoL) in long-term survivors. This study is to investigate the differences in longitudinal recovery of QoL between HID and MSD HSCT using a comprehensive assessment system. Methods: This prospective study enrolled consecutive patients who had received allogenic-HSCT (allo-HSCT) between January 2018 and December 2019 in our center. All patients were informed to complete QoL questionnaires including the Mos 36-Item Short-Form Health Survey (SF-36) and the Functional Assessment of Cancer Therapy Bone Marrow Transplant (FACT-BMT, version 4), using an online applet, before transplantation and at scheduled time points after transplantation. The linear mixed-effects modal was used to analyze the variation trend of different dimensions of both SF-36 and FACT-BMT with different follow-up times. Results: Of the 425 participants, recipients of HID and MSD who survived more than 1 year ( n = 230) were included in the final analysis of QoL (median age [range]: 36, [15,66]). The 3-year overall survival (OS) of HID and MSD was 82.42% and 86.46%, respectively. QoL was assessed using both SF-36 and FACT-BMT and there was longitudinal recovery with clinical significance in the cohort. Compared to MSD-HSCT patients, HID-HSCT recipients demonstrated superior QoL recovery in some subscales describing physical and mental wellness. Specifically, the difference in physical performance is more remarkable using FACT-BMT whereas that in mental wellness is more significant using SF36. In the subsequent stratified analysis, patients with a history of aGVHD or CMV reactivation demonstrated inferior QoL. Conclusions and relevance: Long-term survivors of HID HSCT achieved better QoL in some sub-scales compared to MSD HSCT. In addition, SF-36 and FACT-BMT demonstrated different performance thus combination of both improved capacity of the evaluation system.
Publisher: American Chemical Society (ACS)
Date: 05-06-2018
Publisher: Frontiers Media SA
Date: 06-04-2022
Publisher: Elsevier BV
Date: 2021
DOI: 10.2139/SSRN.3864444
Publisher: BMJ
Date: 06-2020
Abstract: Analysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs. We used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical s les were restriction enzyme digested with two 6-cutters, NcoI and BspHI and the flanking genomic DNA lified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the licons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters. Both inverse PCR and cassette ligation PCR could successfully lify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3′long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing. Our study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce lification bias and improve single nucleotide resolution.
Publisher: Springer Science and Business Media LLC
Date: 22-10-2008
Publisher: Wiley
Date: 08-2010
DOI: 10.1111/J.1399-3062.2010.00497.X
Abstract: Invasive fungal infections (IFI) are frequent complications of allogeneic hematopoietic stem cell transplantation (SCT) and major causes of a poor prognosis. Analysis of risk factors and prognosis of IFI are important for the control of IFI. We retrospectively reviewed the medical records of all allogeneic SCT recipients from January 2000 to December 2007. For the homogeneity of analysis, only 286 patients with no history of IFI were included. Fifty-five cases of IFI were identified, corresponding to cumulative incidence of 19.8%. Different overall survival rates were recorded in patients with or without IFI (20.3% vs. 55.3%, P=0.004). Multivariate analysis identified 2 risk factors of IFI: use of high-dose steroid and cytomegalovirus infections, and risk stratification can classify the patients into 3 subgroups with different incidences of IFI (9.2%, 25.4%, and 34.1%, P=0.0007). Further analysis confirmed use of steroid as a risk factor of inferior prognosis in IFI cases (0 vs. 57.1%, P<0.0001), which was due to higher rates of IFI-related mortality (64.7% vs. 23.9%, P=0.018). Post-transplant IFI was an unfavorable prognostic factor of the SCT recipients, and risk stratification can identify patients with high risk of IFI. Use of steroid played an important role in the pathogenesis as well as prognosis of IFI.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543108.V1
Abstract: Supplementary Figure S1: GVHD survival of lethally-irradiated (day 0, 1000cGy split dose) B6.WT, B6.ASC-/- or B6.NLRP3-/- recipients transplanted with BALB/c BM (5x106) and T cells (5x106). The non-GVHD control group received TCD BM cells (5x106) only. Results combined from 2 independent experiments (n = 20 per T cell-replete group, n = 8 for TCD BM group). ****P 0.0001 B6.NLRP3-/- vs. B6.WT. Results are presented as means {plus minus} SEM.
Publisher: American Society for Clinical Investigation
Date: 03-01-2023
DOI: 10.1172/JCI153733
Publisher: Elsevier BV
Date: 11-2019
Publisher: eLife Sciences Publications, Ltd
Date: 06-07-2015
DOI: 10.7554/ELIFE.06942
Abstract: We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.
Publisher: Frontiers Media SA
Date: 06-08-2019
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550138
Abstract: Abstract The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8 sup + /sup T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8 sup + /sup T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation. /
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543105
Abstract: Supplementary Table S1 (related to Fig. 4A): (A) Ingenuity Pathway Analysis derived from the uncorrected dataset displaying top canonical signaling pathways involving differentially regulated genes in recipients as described in Fig.4A. (B) 10 selected immunologically relevant genes identified as differentially regulated in mice transplanted with B6.WT or B6.ASC-/- grafts.
Publisher: Springer Science and Business Media LLC
Date: 02-09-2015
Publisher: The Royal Society
Date: 09-2016
DOI: 10.1098/RSOB.160183
Abstract: The ability to accurately predict the DNA targets and interacting cofactors of transcriptional regulators from genome-wide data can significantly advance our understanding of gene regulatory networks. NKX2-5 is a homeodomain transcription factor that sits high in the cardiac gene regulatory network and is essential for normal heart development. We previously identified genomic targets for NKX2-5 in mouse HL-1 atrial cardiomyocytes using DNA-adenine methyltransferase identification (DamID). Here, we apply machine learning algorithms and propose a knowledge-based feature selection method for predicting NKX2-5 protein : protein interactions based on motif grammar in genome-wide DNA-binding data. We assessed model performance using leave-one-out cross-validation and a completely independent DamID experiment performed with replicates. In addition to identifying previously described NKX2-5-interacting proteins, including GATA, HAND and TBX family members, a number of novel interactors were identified, with direct protein : protein interactions between NKX2-5 and retinoid X receptor (RXR), paired-related homeobox (PRRX) and Ikaros zinc fingers (IKZF) validated using the yeast two-hybrid assay. We also found that the interaction of RXRα with NKX2-5 mutations found in congenital heart disease (Q187H, R189G and R190H) was altered. These findings highlight an intuitive approach to accessing protein–protein interaction information of transcription factors in DNA-binding experiments.
Publisher: Future Medicine Ltd
Date: 04-2019
Abstract: Aim: Posaconazole (POS) is an extended-spectrum triazole approved for treatment of refractory invasive fungal disease. Outcome analysis of POS salvage therapy is of great clinical significance. Methods: We retrospectively reviewed data for 113 patients who received POS oral suspension as salvage therapy between December 2013 and May 2018. Results: Cumulative success rate at 12 weeks was 53.1% (60/113) with a rapid increase within the first 3–4 weeks and a plateau at 6 weeks after POS initiation. Multivariate logistic regression analysis showed that disseminated infection, prolonged neutropenia, severe graft-versus-host disease and high-dose steroid use were independent risk factors of response. Conclusion: POS oral suspension is effective as a salvage therapy for invasive fungal disease.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-0027
DOI: 10.1158/2326-6066.22543108
Abstract: Supplementary Figure S1: GVHD survival of lethally-irradiated (day 0, 1000cGy split dose) B6.WT, B6.ASC-/- or B6.NLRP3-/- recipients transplanted with BALB/c BM (5x106) and T cells (5x106). The non-GVHD control group received TCD BM cells (5x106) only. Results combined from 2 independent experiments (n = 20 per T cell-replete group, n = 8 for TCD BM group). ****P 0.0001 B6.NLRP3-/- vs. B6.WT. Results are presented as means {plus minus} SEM.
Publisher: American Association for Cancer Research (AACR)
Date: 08-2020
DOI: 10.1158/2326-6066.CIR-19-0653
Abstract: The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.JTCT.2022.06.007
Abstract: Natural killer (NK) cells are the first lymphocyte population to recover after allogenic hematopoietic stem cell transplantation (allo-HSCT) and mediate potent graft versus leukemia effect, particularly in the settings of T-cell depletion. However, the significance of NK cells after unmanipulated transplantation is less clear, and factors affecting early NK reconstitution remain elusive. We retrospectively analyze 180 patients with acute myeloid leukemia or myelodysplastic syndrome who received unmanipulated allografts. We focus on the early NK reconstitution and its association with disease relapse, overall survival, and cytomegalovirus (CMV) reactivation. We also analyze factors that affect NK recovery, such as dose of CD34
Publisher: Elsevier BV
Date: 09-2011
Publisher: Cold Spring Harbor Laboratory
Date: 07-11-2019
DOI: 10.1101/833897
Abstract: Vector integration site analysis can be important in the follow-up of patients who received gene-modified cells, but current platforms based on next-generation sequencing are expensive and relatively inaccessible. We analyzed polyclonal T cells transduced by a gammaretroviral vector, SFG.iCasp9.2A.ΔCD19, from a clinical trial. Following restriction enzyme digestion, the unknown flanking genomic sequences were lified by inverse polymerase chain reaction (PCR) or cassette ligation PCR. Nanopore sequencing could identify thousands of unique integration sites within polyclonal s les, with cassette ligation PCR showing less bias. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis.
Publisher: Elsevier BV
Date: 08-2023
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543105.V1
Abstract: Supplementary Table S1 (related to Fig. 4A): (A) Ingenuity Pathway Analysis derived from the uncorrected dataset displaying top canonical signaling pathways involving differentially regulated genes in recipients as described in Fig.4A. (B) 10 selected immunologically relevant genes identified as differentially regulated in mice transplanted with B6.WT or B6.ASC-/- grafts.
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.JACC.2014.10.069
Abstract: Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein genes, and left ventricular hypertrophy (LVH) develops as an adaptive response to sarcomere dysfunction. It remains unclear whether persistent expression of the mutant gene is required for LVH or whether early gene expression acts as an immutable inductive trigger. The aim of this study was to use a regulatable murine model of HCM to study the reversibility of pathological LVH. The authors generated a double-transgenic mouse model, tTAxαMHCR403Q, in which expression of the HCM-causing Arg403Gln mutation in the α-myosin heavy chain (MHC) gene is inhibited by doxycycline administration. Cardiac structure and function were evaluated in groups of mice that received doxycycline for varying periods from 0 to 40 weeks of age. Untreated tTAxαMHCR403Q mice showed increased left ventricular (LV) mass, contractile dysfunction, myofibrillar disarray, and fibrosis. In contrast, mice treated with doxycycline from conception to 6 weeks had markedly less LVH and fibrosis at 40 weeks. Transgene inhibition from 6 weeks reduced fibrosis but did not prevent LVH or functional changes. There were no differences in LV parameters at 40 weeks between mice with transgene inhibition from 20 weeks and mice with continuous transgene expression. These findings highlight the critical role of the early postnatal period in HCM pathogenesis and suggest that mutant sarcomeres manifest irreversible cardiomyocyte defects that induce LVH. In HCM, mutation-silencing therapies are likely to be ineffective for hypertrophy regression and would have to be administered very early in life to prevent hypertrophy development.
Publisher: American Society for Clinical Investigation
Date: 15-02-2023
DOI: 10.1172/JCI157907
Publisher: American Society of Hematology
Date: 30-12-2021
Abstract: Donor and recipient cytomegalovirus (CMV) serostatus correlate with transplant-related mortality that is associated with reduced survival following allogeneic stem cell transplant (SCT). Prior epidemiologic studies have suggested that CMV seronegative recipients (R–) receiving a CMV-seropositive graft (D+) experience inferior outcomes compared with other serostatus combinations, an observation that appears independent of viral reactivation. We therefore investigated the hypothesis that prior donor CMV exposure irreversibly modifies immunologic function after SCT. We identified a CD4+/CD57+/CD27– T-cell subset that was differentially expressed between D+ and D– transplants and validated results with 120 patient s les. This T-cell subset represents an average of 2.9% (D–/R–), 18% (D–/R+), 12% (D+/R–), and 19.6% (D+/R+) (P & .0001) of the total CD4+ T-cell compartment and stably persists for at least several years post-SCT. Even in the absence of CMV reactivation post-SCT, D+/R– transplants displayed a significant enrichment of these cells compared with D–/R– transplants (P = .0078). These are effector memory cells (CCR7–/CD45RA+/−) that express T-bet, Eomesodermin, granzyme B, secrete Th1 cytokines, and are enriched in CMV-specific T cells. These cells are associated with decreased T-cell receptor ersity (P & .0001) and reduced proportions of major histocompatibility class (MHC) II expressing classical monocytes (P & .0001), myeloid (P = .024), and plasmacytoid dendritic cells (P = .0014). These data describe a highly expanded CD4+ T-cell population and putative mechanisms by which prior donor or recipient CMV exposure may create a lasting immunologic imprint following SCT, providing a rationale for using D– grafts for R– transplant recipients.
Publisher: Cold Spring Harbor Laboratory
Date: 03-10-2014
Publisher: MDPI AG
Date: 09-10-2018
DOI: 10.3390/IJMS19103088
Abstract: There is a pressing need to develop vaccines for schistosomiasis given the current heavy dependency on praziquantel as the only available drug for treatment. We previously showed the ligand domain of the Schistosoma japonicum insulin receptor 1 and 2 (rSjLD1 and 2) fusion proteins conferred solid protection in mice against challenge infection with S. japonicum. To improve vaccine efficacy, we compared the immunogenicity and protective efficacy of rSjLD1 on its own and in combination with S. japonicum triose-phosphate isomerase (SjTPI), formulated with either of two adjuvants (QuilA and montanide ISA 720VG) in murine vaccine trials against S. japonicum challenge. The level of protection was higher in mice vaccinated only with rSjLD1 formulated with either adjuvant rSjTPI or the rSjTPI-rSjLD1 combination resulted in a lower level of protection. Mirroring our previous results, there were significant reductions in the number of female worms (30–44%), faecal eggs (61–68%), liver eggs (44–56%), intestinal eggs (46–48%) and mature intestinal eggs (58–63%) in the rSjLD1-vaccinated mice compared with the adjuvant only groups. At 6-weeks post-cercarial challenge, a significantly increased production of interferon gamma (IFNγ) in rSjLD1-stimulated splenic CD4+ T cells was observed in the rSjLD1-vaccinated mice suggesting a Th1-type response is associated with the generated level of protective efficacy.
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.SMIM.2019.101322
Abstract: Interleukin-10 (IL-10) is a multi-faceted anti-inflammatory cytokine which plays an essential role in immune tolerance. Indeed, deficiency of IL-10 or its receptor results in aberrant immune responses that lead to immunopathology. Graft-versus-host disease (GVHD) is the limiting complication of allogeneic stem cell transplantation (SCT) and results from an imbalance in pathological versus regulatory immune networks. A number of immune cells exert their immunomodulatory role through secretion of IL-10 or induction of IL-10-secreting cells after SCT. Type-1 regulatory T cells (Tr1 cells) and FoxP3
Publisher: American Society for Clinical Investigation
Date: 22-11-2021
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.YGENO.2017.01.006
Abstract: DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion.
Location: China
Location: United States of America
No related grants have been discovered for ping zhang.