A Multicenter Randomized Phase II Study of Single Agent Efficacy and Optimal Combination Sequence of Everolimus and Pasireotide LAR in Advanced Thyroid Cancer.

PURPOSE: Aberrant mTOR pathway and somatostatin receptor signaling are implicated in thyroid cancer and offer potential therapeutic targets. We assessed the clinical efficacy of everolimus and Pasireotide long-acting release (LAR) in radioiodine-refractory differentiated thyroid cancer (DTC) and medullary thyroid cancer (MTC). PATIENTS AND METHODS: Adults with progressive MTC and DTC untreated or treated with no more than one systemic agent were eligible. The trial was designed to establish the most promising regimen and the optimal combination sequence. Patients were randomized to start treatment with single agent everolimus (10 mg QD; Arm A), pasireotide-LAR (60 mg intramuscular injection, Q4 weeks; Arm B), or the combination (Arm C). At initial progression (PFS1), patients on Arm A or B switched to the combination and continued until progression (PFS2). Efficacy was measured by RECIST criteria. RESULTS: Study enrolled 42 patients: median age 65 years; female 17 (40.5%); White 31 (73.8%), African American 6 (14.3%), others 5 (11.9); DTC 32 (76.2%); MTC 10 (23.8%). There was no objective response by RECIST criteria across the three arms. Median and 1-year PFS1 rates were 8.3, 1.8, 8.1 months and 49.9%, 36.4%, 25.0% for Arms A, B, C, respectively. Median and 1-year PFS2 rates were 26.3, 17.5, 8.1 months and 78.4%, 70.0%, 25% for Arms A, B, C, respectively. The most frequent adverse events were anemia, stomatitis, fatigue, hyperglycemia, and hypercholesterolemia. CONCLUSIONS: The combination of everolimus and pasireotide-LAR showed promising efficacy over single agent. The delayed combination of everolimus and pasireotide-LAR following progression on single agent everolimus appeared intriguing as a combination strategy.

Repetitive Transcranial Magnetic Stimulation as a Therapeutic and Probe in Schizophrenia: Examining the Role of Neuroimaging and Future Directions.

Schizophrenia is a complex condition associated with perceptual disturbances, decreased motivation and affect, and disrupted cognition. Individuals living with schizophrenia may experience myriad poor outcomes, including impairment in independent living and function as well as decreased life expectancy. Though existing treatments may offer benefit, many individuals still experience treatment resistant and disabling symptoms. In light of the negative outcomes associated with schizophrenia and the limitations in currently available treatments, there is a significant need for novel therapeutic interventions. Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that can modulate the activity of discrete cortical regions, allowing direct manipulation of local brain activation and indirect manipulation of the target's associated neural networks. rTMS has been studied in schizophrenia for the treatment of auditory hallucinations, negative symptoms, and cognitive deficits, with mixed results. The field's inability to arrive at a consensus on the use rTMS in schizophrenia has stemmed from a variety of issues, perhaps most notably the significant heterogeneity amongst existing trials. In addition, it is likely that factors specific to schizophrenia, rather than the rTMS itself, have presented barriers to the interpretation of existing results. However, advances in approaches to rTMS as a biologic probe and therapeutic, many of which include the integration of neuroimaging with rTMS, offer hope that this technology may still play a role in improving the understanding and treatment of schizophrenia.

Loss of TG-Interacting Factor 1 decreases survival in mouse models of myeloid leukaemia.

TG-Interacting Factor 1 (Tgif1) affects proliferation and differentiation of myeloid cells and regulates self-renewal of haematopoietic stem cells (HSCs). To determine its impact on leukaemic haematopoiesis, we induced acute or chronic myeloid leukaemias (AML or CML) in mice by enforced expression of MLL-AF9 or BCR-ABL, respectively, in Tgif1+/+ or Tgif1-/- haematopoietic stem and progenitor cells (HSPCs) and transplanted them into syngeneic recipients. We find that loss of Tgif1 accelerates leukaemic progression and shortens survival in mice with either AML or CML. Leukaemia-initiating cells (LICs) occur with higher frequency in AML among mice transplanted with MLL-AF9-transduced Tgif1-/- HSPCs than with Tgif1+/+ BMCs. Moreover, AML in mice generated with Tgif1-/- HSPCs are chemotherapy resistant and relapse more rapidly than those whose AML arose in Tgif1+/+ HSPCs. Whole transcriptome analysis shows significant alterations in gene expression profiles associated with transforming growth factor-beta (TGF-beta) and retinoic acid (RA) signalling pathways because of Tgif1 loss. These findings indicate that Tgif1 has a protective role in myeloid leukaemia initiation and progression, and its anti-leukaemic contributions are connected to TGF-beta- and RA-driven functions.

Strategies for Appropriate Selection of SGLT2-i vs. GLP1-RA in Persons with Diabetes and Cardiovascular Disease.

PURPOSE OF REVIEW: This review will serve to highlight the clinical rationale used in the selection of sodium-glucose cotransporter 2 inhibitors (SGLT2-i) or glucagon-like peptide 1 receptor agonists (GLP1-ra). RECENT FINDINGS: SGLT2-i and GLP1-ra are the first anti-hyperglycemics to demonstrate significant cardiovascular benefit in multiple cardiovascular outcomes trials (CVOTs), with benefits that are consistent across class of medication. Diabetes is a major risk factor for morbidity and mortality from cardiovascular disease. Sodium-glucose cotransporter 2 inhibitors (SGLT2-i) and glucagon-like peptide 1 receptor agonists (GLP1-ra) are the first anti-hyperglycemics to demonstrate significant cardiovascular benefit. Given the unique side effect and benefit profiles, appropriate consideration of these agents with a focus on cardiovascular risk reduction requires an individualized approach.

Epo reprograms the epigenome of erythroid cells.

The hormone erythropoietin (Epo) is required for erythropoiesis, yet its molecular mechanism of action remains poorly understood, particularly with respect to chromatin dynamics. To investigate how Epo modulates the erythroid epigenome, we performed epigenetic profiling using an ex vivo murine cell system that undergoes synchronous erythroid maturation in response to Epo stimulation. Our findings define the repertoire of Epo-modulated enhancers, illuminating a new facet of Epo signaling. First, a large number of enhancers rapidly responded to Epo stimulation, revealing a cis-regulatory network of Epo-responsive enhancers. In contrast, most of the other identified enhancers remained in an active acetylated state during Epo signaling, suggesting that most erythroid enhancers are established at an earlier precursor stage. Second, we identified several hundred super-enhancers that were linked to key erythroid genes, such as Tal1, Bcl11a, and Mir144/451. Third, experimental and computational validation revealed that many predicted enhancer regions were occupied by TAL1 and enriched with DNA-binding motifs for GATA1, KLF1, TAL1/E-box, and STAT5. Additionally, many of these cis-regulatory regions were conserved evolutionarily and displayed correlated enhancer:promoter acetylation. Together, these findings define a cis-regulatory enhancer network for Epo signaling during erythropoiesis, and provide the framework for future studies involving the interplay of epigenetics and Epo signaling.

LIM-Only Protein 4 (LMO4) and LIM Domain Binding Protein 1 (LDB1) Promote Growth and Metastasis of Human Head and Neck Cancer (LMO4 and LDB1 in Head and Neck Cancer).

Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.

Requirement for ssbp2 in hematopoietic stem cell maintenance and stress response.

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.

A single nucleotide polymorphism in SLC7A5 is associated with gastrointestinal toxicity after high-dose melphalan and autologous stem cell transplantation for multiple myeloma.

Multiple myeloma is the most frequent indication for high-dose melphalan (HDM) chemotherapy with autologous stem cell transplantation (ASCT). Gastrointestinal symptoms represent the most significant nonhematological toxicity of HDM. However, specific, especially genetic, predictors of their incidence or clinical severity are lacking. The amino acid transporters LAT1 and LAT2 encoded by the SLC7A5 and SLC7A8 genes, respectively, are the principal mediators of melphalan uptake into cells. To determine whether genetic variability at these loci contributed to interindividual differences in the development of gastrointestinal complications of HDM, we analyzed single nucleotide polymorphisms (SNPs) in these genes in 135 patients with multiple myeloma treated with HDM and ASCT and correlated these with the need for total parenteral nutrition (TPN). Seven SNPs in SLC7A5 and 20 in SLC7A8 were genotyped. Multiple analyses indicated that 1 SNP in the first intron of SLC7A5, rs4240803, was significantly associated with TPN use (odds ratio = .45, 95% confidence interval, .25 to .79; P = .007). Further, every haplotype that correlated with TPN requirement included this SNP. These results suggest that variability in melphalan transport affects mucosal injury after HDM. This finding could help in individualizing the dose of this effective and widely used chemotherapeutic agent for multiple myeloma.

Tgif1 regulates quiescence and self-renewal of hematopoietic stem cells.

TG-interacting factor 1 (TGIF1) is a transcriptional repressor that can modulate retinoic acid and transforming growth factor ß signaling pathways. It is required for myeloid progenitor cell differentiation and survival, and mutations in the TGIF1 gene cause holoprosencephaly. Furthermore, we have previously observed that acute myelogenous leukemia (AML) patients with low TGIF1 levels had worse prognoses. Here, we explored the role of Tgif1 in murine hematopoietic stem cell (HSC) function. CFU assays showed that Tgif1(-/-) bone marrow cells produced more total colonies and had higher serial CFU potential. These effects were also observed in vivo, where Tgif1(-/-) bone marrow cells had higher repopulation potential in short- and long-term competitive repopulation assays than wild-type cells. Serial transplantation and replating studies showed that Tgif1(-/-) HSCs exhibited greater self-renewal and were less proliferative and more quiescent than wild-type cells, suggesting that Tgif1 is required for stem cells to enter the cell cycle. Furthermore, HSCs from Tgif1(+/-) mice had a phenotype similar to that of HSCs from Tgif1(-/-) mice, while bone marrow cells with overexpressing Tgif1 showed increased proliferation and lower survival in long-term transplant studies. Taken together, our data suggest that Tgif1 suppresses stem cell self-renewal and provide clues as to how reduced expression of TGIF1 may contribute to poor long-term survival in patients with AML.

Immunomodulatory nonablative conditioning regimen for B-cell lymphoid malignancies.

Twenty-six patients with recurrent CD20(+) B-cell lymphoid malignancies received fludarabine, cyclophosphamide, and rituximab-based nonablative conditioning followed by either matched related (n = 18) or unrelated (n = 8) donor allogeneic stem cell transplantation (allo-SCT) between March 2008 and May 2011. Median age of patients at transplantation was 59 years (range, 41-64 years). At diagnosis, 20 (77%) had stage IV disease; 23 (88%) received ≥3 regimens, 14 (54%) received ≥4 regimens, and 4 (15%) had earlier autologous-SCT. All patients had either chemosensitive or stable disease and nine (35%) were in complete remission before transplantation. At the time of analysis, 17 patients were alive with an estimated 2-year overall survival and progression-free survival rate of 63% and nonrelapse mortality of 25%. Grade II to IV acute graft-vs-host-disease occurred in 8 (31%) and chronic graft-vs-host-disease in 6 (23%) patients (extensive, n = 3). Causes of death include progressive disease in four, acute graft-vs-host-disease in two (both after receiving donor lymphocyte infusion for mixed chimerism with residual disease), infection in one, and other (e.g., substance abuse, leukoencephalopathy) in two. Six patients required rehospitalization within 100 days of SCT (mean = 10 days; range, 3-18 days). Our data support fludarabine, cyclophosphamide, and rituximab-based nonablative conditioning allo-SCT in CD20(+) B-cell lymphoid malignancies and it is time to compare this regimen with an alternative reduced-intensity conditioning regimen in B-cell malignancies.

Consolidative therapy with stem cell transplantation improves survival of patients with mantle cell lymphoma after any induction regimen.

Intensive induction regimen followed by high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) is frequently used to improve outcomes in patients with mantle-cell lymphoma. The comparative impact of conventional vs intensive induction regimen before transplantation is unknown. Forty-eight patients with mantle-cell lymphoma receiving SCT at our institution between January 2000 and December 2010 were included in this study. At the time of initial presentation, 43 (89.5%) had stage IV disease and 18 (37.5%) received more than one chemotherapy regimen before transplantation. Forty patients underwent auto-SCT and 7 had allogeneic SCT (allo-SCT); 1 patient had an allo-SCT for relapsed disease after auto-SCT. At the time of this analysis (median follow-up of 6 years from diagnosis and 4 years from transplantation), 40 patients (88%) were alive with a 5-year disease-free survival of 74.8%. Age, disease stage, number of regimens pre-SCT, pre-SCT disease status, and type of SCT had no impact on long-term outcomes. Importantly, there were no differences among the types of induction regimen on outcomes in this cohort receiving SCT. Based on our data, we believe that future studies should focus on strategies to prevent disease relapse rather than comparing induction regimens before stem cell transplantation.

The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.

Monocytopoiesis involves the stepwise differentiation in the bone marrow (BM) of common myeloid precursors (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and while it had been reported to be expressed by BM-derived macrophages, its role in monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal of the G(1) and S phases. In conjunction, steady-state levels of p16(Ink4a) mRNA were increased and those of Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated the association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during monocytopoiesis and the requirement for direct DNA binding in these processes.

Eto2/MTG16 and MTGR1 are heteromeric corepressors of the TAL1/SCL transcription factor in murine erythroid progenitors.

The TAL1 (or SCL) gene, originally discovered through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. To identify its interaction partners, we expressed a tandem epitope-tagged protein in murine erythroleukemia (MEL) cells and characterized affinity-purified Tal1-containing complexes by liquid chromatography-tandem mass spectrometry analysis. In addition to known interacting proteins, two proteins related to the Eight-Twenty-One (ETO) corepressor, Eto2/Mtg16 and Mtgr1, were identified from the peptide fragments analyzed. Tal1 interaction with Eto2 and Mtgr1 was verified by coimmunoprecipitation analysis in Tal1, Eto2-, and Mtgr1-transfected COS-7 cells, MEL cells expressing V5 epitope-tagged Tal1 protein, and non-transfected MEL cells. Mapping analysis with Gal4 fusion proteins demonstrated a requirement for the bHLH domain of Tal1 and TAF110 domain of Eto2 for their interaction, and transient transfection and glutathione S-transferase pull-down analysis showed that Mtgr1 and Eto2 enhanced the other's association with Tal1. Enforced expression of Eto2 in differentiating MEL cells inhibited the promoter of the Protein 4.2 (P4.2) gene, a direct target of TAL1 in erythroid progenitors, and transduction of Eto2 and Mtgr1 augmented Tal1-mediated gene repression. Finally, chromatin immunoprecipitation analysis revealed that Eto2 occupancy of the P4.2 promoter in MEL cells decreased with differentiation, in parallel with a decline in Eto2 protein abundance. These results identify Eto2 and Mtgr1 as authentic interaction partners of Tal1 and suggest they act as heteromeric corepressors of this bHLH transcription factor during erythroid differentiation.

Transforming growth-interacting factor (TGIF) regulates proliferation and differentiation of human myeloid leukemia cells.

Transforming growth-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer. TGIF is expressed in hematopoietic stem cells and modulates TGF-beta and retinoic acid (RA) signaling, both of which play an important role in hematopoiesis. We recently reported that TGIF's levels correlate inversely with survival in patients with acute myelogenous leukemia. Here we present the first direct evidence of a role for TGIF in myelopoiesis. We used short hairpin RNA interference to define the effects of TGIF knockdown on proliferation and differentiation of myeloid leukemia-derived cell lines. Decreased TGIF expression resulted in reduced proliferation and differentiation and lower expression of CEBPbeta, CEBPepsilon, PU.1 and RUNX1, key myeloid transcription factors. Furthermore, TGF-beta signaling was increased and RA signaling was decreased. Further insights into the molecular basis of TGIF's effects were provided by a genome-wide chromatin immunoprecipitation-based elucidation of TGIF target genes. Together, these data suggest that TGIF has an important role myelopoiesis and may regulate the balance between proliferation and differentiation. Reduced TGIF expression could tip the balance toward quiescence thus providing progenitor as well as hematopoietic stem cells protection from anti-cycle agents.

Histone deacetylase inhibitor romidepsin has differential activity in core binding factor acute myeloid leukemia.

PURPOSE: Recruitment of histone deacetylases (HDAC) is a mechanism of transcriptional repression implicated in the differentiation block in acute myeloid leukemia (AML). We hypothesized that the HDAC inhibitor romidepsin could cause transcriptional derepression, up-regulation of specific target genes in AML, and differentiation of the leukemic clone. The primary objectives of the study were to evaluate the safety and efficacy of romidepsin in advanced AML. EXPERIMENTAL DESIGN: Twenty patients were stratified into cohort A or B based on the absence or presence of chromosomal abnormalities known to recruit HDACs, including those involving core binding factor (CBF). Romidepsin was administered i.v. at 13 mg/m(2)/d on days 1, 8, and 15 of a 28-day cycle. Pharmacodynamic endpoints were evaluated at serial time points. RESULTS: Common adverse effects noted were grade 1 to 2 nausea, anorexia, and fatigue. No objective evidence of antileukemic activity was seen in cohort A. In cohort B, although there were no clinical responses by standard criteria, antileukemic activity was observed in 5 of 7 patients. Two patients had clearance of bone marrow blasts and 3 patients had a >50% decrease in bone marrow blasts. Furthermore, in cohort B, at 24 h, there was a significant increase in MDR1 (P=0.005), p15 (P=0.01), and p14 (P<0.0001) expression. In cohort A, although there was a trend toward up-regulation of MDR1, p15, and p14 expression, these changes were not statistically significant. CONCLUSION: Romidepsin has differential antileukemic and molecular activity in CBF AML. Development of this agent in CBF AML should focus on combinations that target related mechanisms of gene silencing such as DNA methylation.

Single-stranded DNA-binding proteins regulate the abundance and function of the LIM-homeodomain transcription factor LHX2 in pituitary cells.

A family of single-stranded DNA-binding proteins (or SSBPs) has been shown to augment the function of LIM-homeodomain (LIM-HD) transcription factors in embryogenesis by interaction with LIM domain-binding protein-1 (LDB1). No DNA-binding complex has been described, however, containing a LIM-HD protein, LDB1, and SSBP, and the mechanism by which SSBPs affect LIM-HD function had not been elucidated. Through use of electrophoretic mobility shift, antibody supershift, and ChIP analyses, we show that an Lhx2-Ldb1-Ssbp3 complex binds a specific element in the Lhx2 target gene Cga (encoding the alpha subunit of glycoprotein hormones) in the alphaT3-1 pituitary cell line. Using overexpression and knockdown approaches, we demonstrate that SSBP3 inhibits Lhx2 and Ldb1 turnover, stimulates assembly of this DNA-binding complex, promotes its recruitment to the Cga promoter, and enhances Cga transcription. These studies provide novel insights into the regulation of pituitary gene expression and LIM-HD function more generally.

Genomic structure, alternative splicing and expression of TG-interacting factor, in human myeloid leukemia blasts and cell lines.

TG-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer, including leukemias. In this study, we provide the first detailed description of the TGIF locus characterizing 12 TGIF splice isoforms. These isoforms have similar open reading frames but different 5' untranslated regions. TGIF expression data are presented from multiple tissues, cell lines and primary leukemia cells. Isoform-specific real-time PCR analysis showed that even though these isoforms were broadly expressed all except isoform 4, had very low level of expression. In fact, isoform 4 was the predominant TGIF isoform expressed in all tissues analyzed. Since TGIF, levels have recently implicated to play a role in acute myelogenous leukemia we proceeded to characterize the minimal promoter region of isoform 4 as a first step in understanding mechanisms of TGIF expression. As expected for homeobox genes, the minimal promoter region for isoform 4 has multiple Sp1 binding sites and a CpG island raising the possibility that the low TGIF expression seen in some AML patients and leukemia cell lines may be secondary to methylation. Further characterization of expression from this promoter using 5-Aza-2'-deoxycytidine treatment and transient expression assays showed that decreased TGIF expression is likely secondary to active repression and not because of promoter methylation. A detailed characterization of this complex locus is important as it may help to clarify the functions of this gene in brain development and leukemia biology.

A computational model of quantitative chromatin immunoprecipitation (ChIP) analysis.

Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.