Demystifying a methylation mystery at the fetal hemoglobin gene promoter

To solve a problem, sometimes you must wait for technology to catch up. This is what happened with research to modify the epigenetic regulation of hemoglobin genes to treat the blood disorders beta-thalassemia and sickle cell anemia. For decades, scientists have tried to solve the problem of how the body switches hemoglobin genes on and off through 'epigenetic' regulation, a way of changing how genes are expressed without altering the DNA sequence itself. Researchers in the 1980s, including the late Arthur W. Nienhuis, MD, former St. Jude CEO, first tested epigenetic drugs, such as 5-azacytidine, to treat these diseases. By targeting the molecules that regulate gene expression, the scientists turned on  expression of fetal hemoglobin, the form of hemoglobin humans express in utero, in patients’ blood precursor cells.

Fetal hemoglobin can alleviate symptoms of sickle cell anemia and beta thalassemia. Indeed, activation of fetal hemoglobin genes through modification of genomic DNA sequences forms the basis of several gene therapies, including the ongoing SAGES1 clinical trial at St. Jude. But until recently, the epigenetic modification research encountered an important obstacle in patients. Historically, scientists attempted to induce epigenetic changes using drugs that act non-specifically across the entire genome. For example, Nienhuis and others found that 5-azacytidine induced fetal hemoglobin expression in those who were treated. 5-azacytidine is a drug that removes methyl groups, small chemical tags that partially control gene expression, across the entire genome. However, that global demethylation across the genome caused too many toxicities for the approach to be used therapeutically.

For the last 40 years, the connection between DNA methylation and fetal hemoglobin expression has been known, but the mechanistic details of the relationship remained a mystery. This is because the prior research was not specific enough to tell scientists if demethylation at the promoter of gamma globin gene, which makes the protein for fetal hemoglobin, was responsible for the beneficial effects of the epigenetic drug identified in the 1980s. Historically, answering this question was challenging because researchers lacked the tools to target epigenetic marks (including methyl groups) with high specificity. This problem has been largely overcome by the recent development of CRISPR-Cas9-based “epigenome editors” which allow researchers to introduce precise epigenetic changes at specific genes of interest. St. Jude researchers used these remarkable tools to answer a longstanding question in the developmental regulation of fetal hemoglobin, as published in Nature Communications.

“We found that the association between DNA methylation and expression at the gamma globin promoter is causal,” said co-corresponding author Mitchell Weiss, MD, PhD, St. Jude Department of Hematology chair. A promoter is a region of DNA upstream of a gene that brings together the necessary machinery to express the gene. The researchers demonstrated a direct relationship between the presence or absence of methylation at the fetal hemoglobin promoter and its expression, settling the long-standing debate.

In standard genome editing, the Cas9 nuclease is directed to a specific region of DNA by a localizing guide RNA, then it cuts at that site in the genome. A newer technology, termed epigenetic editing, uses a mutant Cas9 that does not cut DNA. Instead, it is attached to one of several enzymes that can create epigenetic modifications, including guide RNA-directed installation or removal of DNA methylation at specific genes. With those sequence-specific epigenetic editors, the group revealed that methylation at six specific cytosine bases in the gamma globin gene promoter in red blood precursor cells inhibited expression. In contrast, demethylation specifically at those sites turned on gene expression. These laboratory results resolved the decades-long mystery using this new technology.

“This started with an old, unfinished story,” said co-first author Ruopeng Feng, PhD, St. Jude Department of Hematology. “Now, we’ve definitively proven that if you specifically methylate the gamma globin promoter, it turns the gene off, while if you remove the methylation, it turns it back on.”

Mastering the maze of methylation

Though Feng was not part of the research in the 1980s, he has worked for a long time on understanding how hemoglobin genes are regulated. Nearly 10 years ago, he performed a CRISPR-Cas9 screen to identify novel genes that inhibit the expression of fetal hemoglobin. He discovered that the loss of UHRF1, a protein that maintains DNA methylation during cell division, increased fetal hemoglobin expression. However, this effect was associated with global demethylation, just like the drugs tested in clinical trials in the 1980s. The exact relationship between gamma globin gene expression and promoter DNA methylation was still unclear, but evidence was mounting that the two had a significant interplay.

In the intervening time, both CRISPR-Cas9 technology and scientists’ understanding of methylation significantly improved. Through a serendipitous conversation with the lab of co-corresponding author Merlin Crossley, PhD, University of New South Wales, Australia, Feng gained access and insight into how to adapt CRISPR-Cas9-based epigenetic editors for studying fetal hemoglobin gene expression. The editors had methylating and demethylating variants that he could adapt to modify the gamma globin locus in cells grown in the lab. Then, he validated the result in red blood cell precursors from healthy human donors.

“Nobody has ever been able to specifically remove methylation on the gamma globin promoter locus in red blood cell progenitors before,” Feng said. “Using this technology, we demethylated the gamma globin promoter, and only that promoter, and induced fetal hemoglobin expression, which, to our knowledge, is the first time anyone has been able to do that in these cells.”

Moving methylation modification to the clinic

Weiss’ lab and others are following up on the study to see how to therapeutically exploit demethylation of the gamma globin locus to increase fetal hemoglobin expression. Feng’s experiments showed that gamma globin promoter demethylation increased fetal hemoglobin from approximately 10% of all hemoglobin to more than 30%, which has potential therapeutic utility.

However, modifying methylation still faces many hurdles before being adopted clinically. Epigenetic editors do not alter the DNA sequence, so the changes introduced into blood-forming hematopoietic stem cells used for conventional gene therapy may not be sustained permanently. The solution to that problem may lie in the development of new technologies. Still, the study provides significant foundational information for all scientists working on DNA methylation in general and the mechanisms of gamma globin gene regulation in particular.

“This paper gives an example of how we used a new technology, epigenetic editing, to answer a long-standing question about the regulation of fetal hemoglobin expression,” Weiss said. “Going forward, we will use epigenetic editors and other genetic tools to investigate how methylation controls the perinatal developmental switch from fetal to adult hemoglobin, and hopefully, we will gain more general insights into gene regulation that could have a positive impact for treating many diseases.”