1. Translated by Wassal Alhammad
KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells Laura J. Norton, Alister P.W. Funnell (2017).Blood Advances 1:
Hi my name is … and l am here to better understanding how KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells
Translated by Wassal Alhammad

2. Hemoglobin (taken from, Quora Website)
Hemoglobin is a protein that is responsible for transporting oxygen from the lung to the various tissues and organs of human body. It composed of Two proteins, alpha and beta. In the center there are heme group which is a compound to hold the iron.
(taken from, Quora Website)

3. Hemoglobin Switching Switching of Hb
 Part of this hemoglobin is expressed from a cassette of genes called human β- locus. This locus contains three different types of genes that makes three different types of hemoglobin: Embryonic, Fetal (HbF), and Adult hemoglobin (HbA). The hemoglobin switching happens when  Embryonic hemoglobin, blue line has 60% of hemoglobin in first month of pregnancy, and drops to 0%, shut down in the 3rd month. Fetal hemoglobin (HbF) (red line) also activates in 1st month and the amount of hemoglobin increases in 4 and 5 month of pregnancy, it start to deactivates before getting birth. while Adult hemoglobin (HbA) (gray line), activates around the 4 month and the switching start in the cross over region, when the Fetal start to deactivates and the adult start to activates/ increases.
Modified from semanticscholar website.

4. Hemoglobin Disorders Sickle cell anemia β-thalassemia
So as the adult hemoglobin can be mutated, this leads to hemoglobin disorder, and will result in sickle cell anemia, which change the shape of red blood cells so it couldn’t function in carrying the oxygen and block the blood flow. These mutations in the hemoglobin gene in chromosome 11 also produce a disease called β-thalassemia.
From CIRMWEB (2015). 

5. Regulation of Hemoglobin Switching
understanding the mechanism of Hb switching is very important to reverse the switching from adult to fetal.
Being able to reverse switching means being able to treat sickle cell anemia and beta-thalassemia.
This study build upon previous finding that Kruppel-like transcription factor 1 (KLF1) is a central protein in many processes related to hemoglobin metabolism and red blood cell development. It was shown that KLF1 regulates the expression of many repressor proteins such as BCL11A. ZBTB7A is another repressor protein that works similar to BCL11A in repressing the production of HbF.
KLF1 increase ZBTB7A expression by binding to its gene promoter (top). ZBTB7A protein pinds to peta-globin promoter and prevent peta-globin gene from expression (bottom)

6. Is the regulation of ZBTB7A by KLF1 is achieved by direct binding of KLF1 to the promoter of ZBTB7A gene?
To understand the mechanism of ZBTB7A regulation, the authors would like to know first whether ZBTB7A is regulated by KLF1 and whether this regulation is achieved by direct binding of KLF1 to the promoter of ZBTB7A gene.
Central question of this study is ……

7. Chromatin Immunoprecipitation (ChIP),Followed by DNA sequencing ChIP-Seq
We answer this question by doing The Chromatin immunoprecipitation ,Followed by DNA sequencing ChIP-Seq experiment, which is used to determine whether a transcription factor interacts with a candidate target gene. For our purpose, ChIP experiment were conducted to see whether ZBTB7A is regulated by direct binding of KLF1 to the promotor of ZBTB7A gene. The experiment was conducted on KLF1-inducible estrogen receptor (K1ER) erythroblast cells. Followed by sequencing of the …..

8. Cross-linking
ChIP-Seq is done in multiple steps, 1st is by cross- linking the DNA to various types of binding proteins by formaldehyde, to glue them together.

9. Cell Lysis and DNA fragmentation
2nd lysating the cells to get free chromatin, DNA bound to proteins. By using altra-sound to break down DNA-bound proteins to small fragments.

10. Chromatin immunoprecipitation and elution of DNA
3rd we add antibodies specific to KLF1 to distinguish them from other DNA-binding proteins and precipitated them, then unbind the AG-Ab interaction by elution to get DNA- bound to KLF1

11. Cross-link reversal and DNA purification
 free up the crosslinked DNA by reverse the cross-link, this will get free DNA and proteins , purifying this to get rid of all proteins and antibodies and free DNA and then sequence it to get the result

12. KLF1 binding site in ZBTB7A promoter region
The sequence data of the sample we do chip-seq in
What is a peak? It is the number of DNA copies that were found in this region of the genome
What is the Y-axis in panels D and E? the numbers (left) represent the length of the promoter, 0 is the first nucleotide and 290 is the last, while K1ER is two samples of KLF1-inducible estrogen receptor (K1ER) erythroblast cells and got the same results.
How can you claim that KLF1 regulates ZBTB7A? By showing here the peaks means where KLF1 bind to Erythroid promoter of ZBTB7A
What is the X-axis of panels D and E? , while RefSeq (bottom) mean the start of RNA transcription, the 5.2 kb is a number of the length of the ZBTB7A gene

13. Is the regulation of ZBTB7A by KLF1 is achieved by direct binding of KLF1 to the promoter of ZBTB7A gene?
Using ChIP-Seq, this article showed that KLF1 binds strongly to ZBTB7A promoter in K1ER. Their findings tell us that KLF1 regulate ZBTB7A by direct binding. This discovery could reduce Sickle-cell anemia and β-thalassemia diseases by altering the expression of KLF1 which will alter the expression of ZBTB7A.