beta globin gene cluster

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