scholarly journals GATA-1 Deacetylation and Interaction with HDAC1 Is Critical for GATA-1 Mediated Gene Transcription

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 945-945
Author(s):  
Bowen Yan ◽  
Suming Huang ◽  
Yi Qiu
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Globin Gene ◽  
Gene Expression Pattern ◽  
Direct Interaction ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
Erythroid Progenitor

Abstract The transcription factor GATA-1 is an essential factor for globin gene transcrption and is required for erythroid and megakaryocytic cell differentiation and maturation. GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 chromatin binding activity. However, it is not clear whether GATA-1 acetylation can be reversed by a deacetylase. It is showed that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) associated NURD corepressor complexes through binding to FOG-1. However, we found that the NURD complex does not deacetylate GATA-1. We discovered that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for the interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) blocks the interaction of HDAC1, but doesn't affect its DNA binding in vitro. Importantly, the mutation does not affect the interaction with FOG-1, indicating that GATA-1 direct interaction and indirect association via FOG-1 with HDAC1 are separate events. To further understand the role of the HDAC1-GATA-1 direct interaction in gene transcription and erythropoiesis, we introduced the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Interestingly, upon estradiol induction, GATA-1 2RA does not promote b-globin gene transcription and erythroid differentiation of G1E cells, although GATA-1 2RA is highly acetylated. Chromatin immunoprecipitation assay (ChIP) shows that GATA-1 2RA binds poorly to HS3 and β-globin promoter. Similar binding defect is also detected on GATA-1 promoter, indicating defective GATA-1 recruitment on chromatin. Interestingly, HDAC1 binding to these regions are also significantly reduced, suggesting HDAC1-GATA-1 interaction may be important for GATA-1 deacetylation, as well as stabilizing GATA-1 binding. In order to investigate the effect of GATA-1 2RA in vivo, we generated a GATA-1 2RA knock in mice. The knock in mice are viable but suffered from anemia and thrombocytopenia. β-globin expression reduced at least 50% in knock in mice compare to wild type litter mate. To further identify and compare gene expression profiles regulated by HDAC1 direct or indirect associated GATA-1 during erythroid differentiation, we performed RNA sequencing assays to study the effects of GATA-1 2RA in gene expression in comparison with wild type GATA-1 or GATA-1-V205M (a mutation abolished binding with FOG-1). Expression of GATA-1 2RA largely affects gene expression profile in both GATA-1 activated and repressed genes compared to cells expressing wild type GATA-1. The gene expression pattern in 2RA cells also is largely different from cells expressing GATA-1 V205M, indicating the direct and indirect interaction with HDAC1 may mediate differential functions. Our results indicate that HDAC1 is required for GATA-1 recruitment and GATA-1 mediated transcription regulation. Thus, this study unveils a novel regulation of GATA-1 by its direct interaction with HDAC1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Histone Deacetylase 6 (HDAC6) Regulates Gene Transcription and Enucleation during Erythroid Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 447-447
Author(s):  
Xuehui Li ◽  
Yurong Yang ◽  
Jianrong Lu ◽  
Timothy McKinsey ◽  
Peng Ji ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Gene Transcription ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Regulation Of Transcription ◽  
Actin Ring ◽  
Pol Ii

Abstract The deacetylation of histone and non-histone proteins by histone deacetylase (HDACs) plays a critical role in gene transcription and many other cellular processes in eukaryotic cells. Class II deacetylase HDAC6 is mainly localized in the cytoplasm and deacetylates tubulin and other cytoplasmic proteins. Earlier studies from our laboratory showed that HDAC6 can be transported to the nucleus, and can interact and deacetylate nuclear proteins, such as histones. In this study, we investigate the function of HDAC6 during erythroid differentiation. In proerythroblast cells, HDAC6 is detected in the nucleus and gradually migrates to the cytoplasm upon the induction of differentiation, indicating different HDAC6 functions during erythropoiesis. Inhibition of HDAC6 in mouse fetal liver erythroblasts impairs differentiation and enucleation. β-globin gene transcription is reduced by HDAC6 specific inhibitor, Tubastatin A, in cultured fetal erythroblasts. HDAC6 knockout mice also showed impaired globin gene transcription. HDAC6 specifically interacts with serine 2 phosphorylated Pol II and is recruited mainly at the transcribed region of β-globin, correlating with RNA Pol II recruitment. These results suggest that HDAC6 promotes erythroblast differentiation through the regulation of transcription elongation. HDAC6 also plays a role in regulating the enucleation process during the late stage of erythropoiesis. The formation of the contractile actin ring is disrupted and the enucleation process is blocked in cultured mouse fetal erythroblasts treated with the HDAC6 inhibitor. We further investigated the molecular mechanism of HDAC6 regulated enucleation. We found that HDAC6 interacts with and deacetylates mDia2, an effector of Rho GTPases that is required for erythroblast enucleation. Deacetylation of mDia2 is required for the formation of the contractile actin ring and subsequent enucleation. Altogether, our results identify the important role of HDAC6 in regulating transcription and enucleation during the different stages of mammalian erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Expression Profile of PIP Kinases During In Vitro Human Erythropoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2079-2079
Author(s):  
Tânia Regina Zaccariotto ◽  
Carolina Lanaro ◽  
Dulcinéia Martins Albuquerque ◽  
Magnun N N Santos ◽  
Marcos André Cavalcanti Bezerra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Expression Profile ◽  
Expression Profiles ◽  
Globin Gene ◽  
Gene Expression Profiles ◽  
Erythroid Differentiation ◽  
Globin Genes ◽  
Hb H Disease

Abstract Abstract 2079 Phosphatidylinositol phosphate kinases (PIPKs) are a family of lipid kinase enzymes that produce the second messenger PI4,5P2 (phosphatidylinositol 4,5-biphosphate), which plays an important role in the regulation of a variety of cellular activities, including gene expression. PIPKs are classified into 3 subfamilies — PIPK I (a, b, g), PIPK II (a, b, g) and PIPK III — which are functionally distinct and are located in different subcellular compartments. In a recent study in our laboratory, the PIPKIIa gene was differentially expressed in reticulocytes from 2 siblings with hemoglobin (Hb) H disease who had the same genotype (-a3.7/–SEA). Expression of both the PIPKIIa and b-globin genes were higher in the patient with the higher Hb H level, suggesting a possible relationship between PIPKIIa and the production of globins, particularly b-globin. In light of these findings, the aim of this study was to determine the gene expression profiles of PIPKs (I and II - with their isoforms a, b and g - and III) during erythropoiesis in peripheral blood hematopoietic CD34+ cell culture from 11 healthy volunteers and 6 patients with hemoglobinopathies [2 with a-thalassemia (Hb H disease), 2 with b-thalassemia (homozygous for the IVS-I-6-T-C mutation) and 2 with sickle cell anemia] using quantitative real time PCR (qRT-PCR) and to compare these profiles with the gene expression profiles of a-, b- and g-globins on the 7th, 10th and 13th days of the erythroid culture. In the cell culture from the normal group, expression of the PIPKIIa and other PIPK genes increased during erythroid differentiation, coinciding with the expression profiles of globin genes and showing in particular that a-globin has a significant effect on PIPKIIa (p<0.0001), as the PIPKIIa on a-globin gene (p=0.0002). In the patients, the expression profile of the PIPKIIa gene also increased during differentiation, whereas the results for the other PIPK genes varied. However, mRNA levels differed between patients, indicating greater complexity in individuals with hemoglobinopathies. PIPKIIa expression level was elevated in the culture from one of the a-thalassemia patients (approximately 12 times higher than in the corresponding control) but was lower than the control in one of the b-thalassemia patients. Expression levels of this gene also varied among sickle cell patients. This is the first study of the gene expression profiles of these kinases during in vitro human erythropoiesis. We identified a standard pattern of gene expression for PIPKs, and PIPKIIa in particular, a gradual increase in expression during erythroid differentiation, similar to the pattern for globin genes. This suggests that PI4,5P2, as an important secondary messenger involved in the regulation of gene expression, may play an important role in the regulation of globin gene expression and the normal process of Hb synthesis in red blood cells. Although our results varied between patients, highlighting the complexity of the regulatory systems involved in Hb production, they reinforce the hypothesis of a relationship between PIPKIIa and globin expression. This work was supported by FAPESP, CNPq and CAPES. Disclosures: No relevant conflicts of interest to declare.

Frequent BRAF V600E Mutations Are Identified in CD207+ Cells in LCH Lesions, but BRAF Status does not Correlate with Clinical Presentation of Patients or Transcriptional Profiles of CD207+ Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1372-1372 ◽  
Author(s):  
Tricia L Peters ◽  
Tsz-Kwong Chris Man ◽  
Jeremy Price ◽  
Renelle George ◽  
Phaik Har Lim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Biological Significance ◽  
Braf V600e ◽  
Cell Of Origin ◽  
Wild Type ◽  
Braf Mutations ◽  
Clinical Difference ◽  
The Impact

Abstract Abstract 1372 Background: Very little is known about the cell of origin or the pathogenesis of LCH. There remains debate regarding LCH as a malignant disorder or the result of immune dysregulation. While multiple studies in the past failed to identify significant genetic lesions, an activating mutation (V600E) in the serine/threonine kinase BRAF was recently described in LCH biopsy samples (Badalian-Very et al., 2010). Objective: This study was designed to evaluate the frequency of BRAF mutations in LCH lesions, to identify the cells within the lesions carrying the mutation, and to evaluate the clinical and biological significance of the mutation. Design/Methods: Fresh LCH biopsy samples were collected, cells were sorted into CD3+ and CD207+ fractions, and RNA was purified then amplified into cDNA. Sanger sequencing as well as BRAF allele-specific PCR were performed for each sample. Categorical clinical data was compared to BRAF genotype to evaluate clinical significance of the mutation. Transcriptomes of CD207+ cells were also compared (wild-type BRAF vs V600E) with the Affymetrix U133Plus2.0 platform to determine the impact of the BRAF mutation on global gene expression. Results: The BRAF V600E mutation was consistently identified in cDNA generated from CD207+ cells in 17 of 32 (52%) LCH biopsy samples. Only the wild-type allele was detected in purified T (CD3+) cells from LCH lesions, control epidermal (CD207+) Langerhans cells, and control tonsil T (CD3+) cells. In two cases of recurrent disease, BRAF status was consistent in the presenting and the relapse CD207+ cells: wild-type BRAF in one case and V600E BRAF in another. However, mutation status did not correlate significantly with age (p=0.6), single lesion vs multifocal/systemic (p=1.0), or future recurrent/refractory disease (p=0.2) in this series. Furthermore, unsupervised clustering gene expression profiles CD207+ cells (wild-type BRAF vs V600E) did not segregate datasets based on BRAF status. Using standard statistical analysis, there were no genes identified as significantly up- or down-regulated as a result of the V600E mutation. Conclusion: The BRAF V600E point mutation is the first reproducible molecular abnormality identified in LCH. In this study, we validate the observation that it occurs with high frequency, and definitively localize the pathologic CD207+ cell as the source of the mutation in LCH lesions. Interestingly, while the frequency of the mutation implies some functional significance, in this series there is no statistically significant clinical difference between patients with wild-type or mutated BRAF lesions, and the transcriptomes of LCH CD207+ cells with wild-type and V600E BRAF are indistinguishable. It is possible that the mutation affects LCH pathogenesis at earlier stages in tumorigenesis, or there may be other routes of Ras pathway activation in LCH lesions with wild-type BRAF. While the role for BRAF in LCH pathogenesis remains to be defined, this is an important molecular foothold from which to investigate the biology of LCH. Disclosures: No relevant conflicts of interest to declare.

Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 962-962
Author(s):  
Kenneth R Peterson ◽  
Levi C Makala ◽  
Mayuko Takezaki ◽  
Carlos F Barbas ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Sickle Cell ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Proximal Promoter ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Globin Gene Expression

Abstract A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. Disclosures Makala: Georgia Regents University: Employment.

SATB1 family protein expressed during early erythroid differentiation modifies globin gene expression

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3330-3339 ◽  
Author(s):  
Jie Wen ◽  
Suming Huang ◽  
Heather Rogers ◽  
Liliane A. Dickinson ◽  
Terumi Kohwi-Shigematsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Family Protein ◽  
Globin Promoter ◽  
Globin Gene Expression

AbstractSpecial AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with ϵ-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases γ-globin and down-regulates SATB1 family protein and ϵ-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the β-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the ϵ-globin promoter. SATB1 overexpression increases ϵ-globin and decreases γ-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the ϵ-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the γ-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in ϵ-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the ϵ-globin gene by interaction with specific sites in the β-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation. (Blood. 2005;105:3330-3339)

HDAC1 Can Deacetylate GATA-1 and Regulates Its Activity through a FOG-1 Independent Manner

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5125-5125
Author(s):  
Bowen Yan ◽  
Tao Yang ◽  
Suming Huang ◽  
Yi Qiu
Keyword(s):  
Conflicts Of Interest ◽  
Direct Interaction ◽  
Binding Activity ◽  
Wild Type ◽  
Independent Manner ◽  
Erythroid Progenitor ◽  
Histone Deacetylase 1 ◽  
Dna Binding Activity ◽  
A Genome

Abstract The transcription factor GATA-1 is essential for erythroid and megakaryocytic cell differentiation and maturation. It has been well documented that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) containing NuRD corepressor complexes through an association with FOG-1. Our previous work also shows that HDAC1 acetylation modulates the activity of FOG-1 associated NuRD complexes. Earlier studies show GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 binding activity to chromatin. However, it is not clear whether the acetylation can be reversed by a deacetylase. In this study, we found that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in the deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for its interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) can largely decrease the interaction of these two proteins, but doesn't affect its interaction with FOG-1, indicating that the direct interactions with HDAC1 and FOG-1 dependent association of NuRD complexes are separate events. The mutations also do not affect GATA-1 DNA binding activity in vitro. To further investigate the function of this interaction in erythropoiesis, we introduced wild type or the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Upon estradiol induction, GATA-1 2RA inhibits the differentiation in G1E cells. We performed RNA sequencing to study the effect of GATA-1 2RA in gene expression in comparison with wild type GATA-1 and GATA-1-V205M (a mutation abolished binding with FOG-1) on a genome wild scale. GATA-1 2RA affects GATA-1 function in both GATA-1 activated and repressed genes. Although there is some overlap between 2RA and V205M mutations in both activated and repressed genes, more genes affected by these two mutations are different. Thus, this study unveils a novel regulation for GATA-1 by its direct interaction with HDAC1 during hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Gene expression pattern of chicken erythrocyte nuclei in heterokaryons

1990 ◽  
Vol 97 (1) ◽  
pp. 167-175
Author(s):  
M. Bergman ◽  
N. Ringertz
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Globin Gene ◽  
Gene Expression Pattern ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Strong Expression

Expression of erythro-specific chick genes was studied in heterokaryons prepared by fusing chick erythrocytes (CE) with rat myoblasts. In this type of heterokaryon the inactive erythrocyte nucleus takes up nuclear proteins of myoblast origin and undergoes transcriptional reactivation. In order to study the stability of the genetic programming of the reactivated CE nucleus, chick gene expression was examined by analysis of RNA from the heterokaryons. Probes for several erythro- and chick-specific genes were used. The heterokaryons showed strong expression of the chick histone H5 and adult alpha-globin genes, while other genes, e.g. the transcription factor Eryf1 gene, normally expressed during erythroid differentiation, were not transcribed. Although the CE used were of the definitive lineage, the heterokaryons showed activation of the chick embryonic beta-globin gene, i.e. a gene normally expressed only in CE of the primitive lineage. We conclude that the reactivation of the mature CE nucleus in a rat cytoplasm resulted in a more immature erythroid gene expression pattern. The activation of the embryonic beta-globin gene indicated a switch of the lineage-specific gene expression pattern. This switch occurred in the absence of DNA replication. The strong expression of the globin and H5 genes in heterokaryons, in the absence of expression of the regulatory factor Eryf1, suggested the existence of Eryf1-independent regulatory mechanisms for erythroid gene expression in these cells.

Role of heat shock transcription factor in yeast metallothionein gene expression

1991 ◽  
Vol 11 (7) ◽  
pp. 3676-3681
Author(s):  
W M Yang ◽  
W Gahl ◽  
D Hamer
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Heat Shock ◽  
Gene Transcription ◽  
Heat Shock Factor ◽  
Heat Shock Transcription Factor ◽  
Wild Type ◽  
Promoter Sequences ◽  
Metallothionein Gene ◽  
Upstream Promoter

The induction of Saccharomyces cerevisiae metallothionein gene transcription by Cu and Ag is mediated by the ACE1 transcription factor. In an effort to detect additional stimuli and factors that regulate metallothionein gene transcription, we isolated a Cu-resistant suppressor mutant of an ACE1 deletion strain. Even in the absence of metals, the suppressor mutant exhibited high basal levels of metallothionein gene transcription that required upstream promoter sequences. The suppressor gene was cloned, and its predicted product was shown to correspond to yeast heat shock transcription factor with a single-amino-acid substitution in the DNA-binding domain. The mutant heat shock factor bound strongly to metallothionein gene upstream promoter sequences, whereas wild-type heat shock factor interacted weakly with the same region. Heat treatment led to a slight but reproducible induction of metallothionein gene expression in both wild-type and suppressor strains, and Cd induced transcription in the mutant strain. These studies provide evidence for multiple pathways of metallothionein gene transcriptional regulation in S. cerevisiae.

Transduction of mouse retina by insect cell packaged recombinant adeno-associated virusess and their mutants via intravitreal injection

2021 ◽  
Author(s):  
Zheng Wei ◽  
Xiaomei Liu ◽  
Taiming Li ◽  
Xiaofang Li ◽  
Qungang Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Gene Expression Profiles ◽  
Mouse Retina ◽  
Transduction Efficiency ◽  
Egfp Expression ◽  
Wild Type ◽  
Gene Therapy Vector

Aim: Adeno-associated virus (AAV) is the most preferred gene therapy vector. The purpose of our research is to compare the infection tropism and gene expression efficiency of vitreous injection of recombinant AAVs (rAAVs) and their capsid mutants in mouse retina. Materials & methods: We packaged wild-type rAAV2/2,6,8,9 and their capsid mutants carrying EGFP expression cassette using insect cells. The gene expression profiles of rAAVs and their mutants in mouse retina were evaluated by optical imaging of retinal tissue flat mount and cryosections. Results & conclusion: The results showed that rAAV2 and rAAV2-Y444F mainly targeted retinal ganglion cell; rAAV8, rAAV8-Y733F, rAAV9 and mutants had obvious EGFP expression in retinal pigment epithelium cells. Compared with the wild-type rAAVs, capsid mutants have an improved transduction efficiency in mouse retina cells.

scholarly journals H3 K79 dimethylation marks developmental activation of the β-globin gene but is reduced upon LCR-mediated high-level transcription

Blood ◽  
2008 ◽  
Vol 112 (2) ◽  
pp. 406-414 ◽  
Author(s):  
Tomoyuki Sawado ◽  
Jessica Halow ◽  
Hogune Im ◽  
Tobias Ragoczy ◽  
Emery H. Bresnick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Genome Wide ◽  
Activation Of Transcription ◽  
High Level ◽  
The Relationship ◽  
Mutant Genes ◽  
Gene Expression Levels

Abstract Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired β-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Δ locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the β-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and ΔLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the ΔLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with β-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level β-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

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