In The Name of God
Subject : Beta Globin gene cluster
Alireza Kazemi
Hemoglobin Heme = Protoporphirin +
Hemoglobin Alpha Globin Beta
Globins 16p13.3 Alpha Cluster Telomeric end 30 kb Globins 11p15.5 Beta Cluster near the Telomeric end 70kb
Human Globin Genes
3 Exons (coding) IVS-1 Short (120-130bp) 2 Introns (Non-coding) IVS-2 Long (850-900bp)
Promoter and UTR Enhancer Silencer
Exon Function Exon1 Removing Globin mRNA from cell nuclear
Exon2 Junction to Heme
Exon3 Junction to other globins
Hemoglobin Types
Transcription Factors for Globin synthesis
AP-1,SP-1
General YY-1,USF SCL,TAL-1
TFs
LCR-F1 , EKLF , ATXR Specific GATA-1 , FOG NFE 2 , Nrf 1 , Nrf 2
Regulatory regions in globin genes
1) Basal Promoter +32 to -37
2) Upstream Regulators (UR) -40 to -250
3) Proximal Enhancers in 3’ end of β and γ globin
genes
4) Master Regulators Alpha cluster HS
(MCS-R)
Beta cluster β-LCRs
Basal Promoter in Beta Globin Gene
TFIIBBeta-DRF TBP
TFIID (TAF)RNA Polymerase II
Mutation in Promoter Stop Expression β -Thalassemia
-31 +1 +27 +32
BRE
TATA Inr MTEDPE
-37
Sequence :
Upstream Regulators (UR) in Beta Globin
genes
-250
-40
GATA-1 EKLF
NF-YCP-1
β -DRFTFIIB
BP2bs
NF1bs
GATABB1b
sCAC
CCAC
C
CCAAT β -DRE
β -DRE
Enhancers
Proximal = 3’ end enhancers in β and γ globin genes
Enhancers
Distal = Master regulators : LCR and HS
Proximal Enhancers at 3’ end of Beta and
Gama globin Genes
HBB :
HBG1:
Mutation in
Enhancers
β -ThalassemiaDown expression
γPE
LCR Locus control regions (LCR) are operationally defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number-dependent manner at ectopic chromatin sites .
β-LCRs β-LCRs containing five areas that are sensitive
to DNAse1 erythroid
SpecificDnase1Hypersensitive
Site (eDHS)
HS-1
HS-2
eDHS HS-3
HS-4
HS-5
β-LCRs HS-2 The most important area in
increasing the globin chain synthesis
HS-3 The most important area in
remodelling and opening chromatin structure
LCR Motifs Each of the LCRs have a different number of
these sequences :
GATA GATA-1
CACC SP-1 and EKLF
LCR Motifs MARE
E-BOX SCL(TAL-1)
YY1bs
SP1bs
MARE Sequence Sequence : TGAGTCA
Has an inhibitory and silencing roles in beta
globin transcription
Junction to basic leucine zipper factors AP-
1
(bZIP)
NF-E2
Bach-1
Nrf-1/LCRF1
MARE Sequence Function
In Lower Heme levels In Higher Heme levels
Bach1 + Small maf NF-E2/Nrf2 + Small maf (P45) (P18) Junction to Enhancers Junction to MARE sequence Globin expression
Inhibition of Globin synthesis Hemoglobin
HRI kinase
GATA-1
Functions :
Regulation of globin synthesis
Regulation of Heme synthesis
Regulation of other erythroid transcription
factors
Regulation of hematopoiesis ( MKs , Eo , … )
Regulation of globin synthesis By GATA-1
Junction to 5’ end Silencer in ε genes Down expression
of ε gene Hb Gower
Junction to γ Glycine Silencer Down expression of γ
gene
(HbF )
Intraction with other TFs (e.g. GATA-2 , FOG , EKLF ,
P300/CEP and PU-1)
GATA-1 Connect to multiple sequences of the promoter
and Enhancer in beta globin gene cluster
Loop Formation
increase β-Globin gene expression
GATA-1
GATA-1
GATA-1 P300/CEP
GATA-1 acetylationHistone acetylation
Increase access to Beta globin gene
EKLF Erythroid Krueppel-Like Factor
Functions 1. Chromatin remodeling
2. Modulation of the gamma to beta
globin switch
3. Transcriptional activation
1. EKLF
Three Forms 2. Fetal form (FKLF)
3. Basic form(BKLF)
EKLF
• Globin expression In Adult(α & β) EKLF
In Embryo & Fetal FKLF
BKLF
• EKLF BKLF
• EKLF Mutation BKLF & Globin
EKLF Over expression Thrombocytopenia
Early switch γ to
β
NF-E2
• Consists of two components :
1. P45 Nrf 1,2,3
Bach 1,2
2. P18 Small maf (maf-k)
• Functions Globin synthesis
Heme synthesis
ferrochelatase
PBG
Deaminase
P45 Functions Junction to CEP/P300 , Ubiquitin ligase , TAFII-
130
Open chromatin structure of target genes
Increase access to TFs (e.g. GATA-1, EKLF, SCL)
Globin expression
Inhibition of Methyltransferase MLL2
(myeloid/lymphoid or mixed-lineage leukemia protein 2 )
Switching to HbA• In ε & γ genes UR :
2 Motifs Enhancer SSE(Stage Selector
Element)
Silencer DR-1
DR-1 DRED
Switching to HbA
• In β-Globin regulatory elements :
11 GATA motifs GATA
The sharp increase
β-Globin gene
expression
4 CACC-box EKLF
Drug effects• γ - Aminobutyric acid(GABA) Butyrate Phenylbutyrate HDAC & P300 inhibition Valproic acid Histone Acetylation HbF
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