1. In The Name of God
Subject : Beta Globin gene cluster
Alireza Kazemi

2. Hemoglobin
Heme = Protoporphirin + 𝑭𝒆 𝟐+
Hemoglobin
Alpha
Globin
Beta

3. Globins
16p13.3
Alpha Cluster Telomeric end
30 kb
Globins
11p15.5
Beta Cluster near the Telomeric end
70kb

4. Human Globin Genes
3 Exons (coding)
IVS-1 Short (120-130bp)
2 Introns (Non-coding)
IVS-2 Long (850-900bp)
Promoter
𝟑′
and 𝟓′
UTR Enhancer
Silencer

5. Exon Function
Exon1 Removing Globin mRNA from cell nuclear
Exon2 Junction to Heme
Exon3 Junction to other globins

8. Hemoglobin Types

10. 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

11. 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

12. Basal Promoter in Beta Globin Gene
TFIIB
Beta-DRF TBP
TFIID (TAF)
RNA Polymerase II
Mutation in
Promoter Stop Expression β -Thalassemia
-31 +1 +27 +32
BRE TATA Inr MTE DPE
-37
Sequence :

13. Upstream Regulators (UR) in Beta Globin genes
-250
-40
GATA-1 EKLF
NF-Y
CP-1
β -DRF
TFIIB
BP2bs NF1bs GATA BB1bs CACC CACC
CCAAT β -DRE β -DRE

14. Enhancers
Proximal = 3’ end enhancers in β and
γ globin genes
Enhancers
Distal = Master regulators : LCR and HS

15. Proximal Enhancers at 3’ end of Beta and Gama globin
Genes
HBB :
HBG1:
Mutation in
Enhancers
β -ThalassemiaDown expression
γPE

16. 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 .

17. 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

18. 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

20. 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

21. 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

22. 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

24. HRI kinase

25. GATA-1
 Functions :
 Regulation of globin synthesis
 Regulation of Heme synthesis
 Regulation of other erythroid transcription factors
 Regulation of hematopoiesis ( MKs , Eo , … )

26. 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)

27. GATA-1
Connect to multiple sequences of the promoter and
Enhancer in beta globin gene cluster
Loop Formation
increase β-Globin gene expression

28. GATA-1

29. GATA-1
GATA-1 P300/CEP
GATA-1 acetylation
Histone acetylation
Increase access to Beta globin gene

30. 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)

31. 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

32. 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

33. 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 )

35. Switching to HbA
• In ε & γ genes UR :
2 Motifs Enhancer SSE(Stage Selector Element)
Silencer DR-1
DR-1 DRED

36. Switching to HbA
• In β-Globin regulatory elements :
11 GATA motifs GATA
The sharp increase
β-Globin gene expression
4 CACC-box EKLF

37. Drug effects
• γ - Aminobutyric acid(GABA)
Butyrate
Phenylbutyrate HDAC & P300 inhibition
Valproic acid
Histone Acetylation
HbF