Oxygen sensing – an essential process for survival

The researchers’ findings touch a tremendous array of biological processes, and the details they have unearthed present possible strategies for revving up or reining in these activities. Such ventures might lead toward new therapeutics for a wide range of disorders, including some types of anemia, cardiovascular disease, and cancer.

Energizing hypoxic genes

Animals require oxygen to extract energy from food, but too much of the chemical creates peril, as certain oxygen-containing compounds wreak molecular havoc. To handle this challenge, organisms have evolved honed systems to furnish optimal supplies. For example, many creatures use red blood cells to transport oxygen to tissues deep within their bodies. When its concentration sags, the body generates the hormone erythropoietin, which boosts red blood-cell production. 

In the early 1990s, Semenza and Ratcliffe were trying to understand how oxygen deprivation—hypoxia—triggers the erythropoietin gene. They and others identified a DNA sequence that is necessary for hypoxia-dependent activation. Placement of this DNA stretch next to other genes renders those genes inducible by low-oxygen conditions too. Semenza showed that protein from the nucleus sticks to this DNA-control region, but only when oxygen is scarce. Furthermore, sequence alterations that eliminate protein binding to DNA also obliterate hypoxia-spurred gene stimulation. Perhaps, he proposed, the nuclear protein, which he called Hypoxia-Inducible Factor 1 (HIF-1), sits down on DNA when cells lack oxygen and rouses adjacent genes. 

Semenza and postdoctoral fellow Guang Wang achieved a major breakthrough in 1995, when they purified HIF-1 and found that it contains two protein partners, HIF-1α and HIF-1β.  The HIF-1α component was novel, and they isolated its gene from human cells. HIF-1α vanished quickly when Semenza shifted cells from low- to high-oxygen conditions. 

A profusion of HIF-1-driven genes 

Erythropoietin in adults is produced predominantly by specialized kidney cells, and scientists had long thought that mechanisms governing its manufacture were peculiar to those cells. With HIF-1 and its binding sequences in hand, Ratcliffe and Semenza upended this idea.

In complementary work, the scientists showed that hypoxia provokes HIF-1 to bind and activate target genes in many mammalian cells that do not make erythropoietin. Their observations suggested that multiple kinds of cells use HIF-1 to goad numerous genes in response to low-oxygen conditions.

Semenza and Ratcliffe subsequently confirmed this idea, and the list of hypoxia-induced genes expanded rapidly. In 1996, for instance, Semenza demonstrated that HIF-1 activates the gene for a key participant in blood-vessel formation, vascular endothelial growth factor (VEGF). This result extended HIF-1 into another key system by which the body can augment oxygen delivery. These and other findings established that HIF-1 lies at the core of an elaborate physiological network that ensures advantageous responses to oxygen.

Smothering HIF-1

The observation that the amount of HIF-1 plummets when cells are shifted to high-oxygen conditions squared with the factor’s hallmark ability to activate target genes only when oxygen is limited. It also raised a crucial question: What drives HIF-1 destruction? The answer came from an unexpected direction. 

A familial cancer syndrome called von Hippel-Lindau (VHL) disease owes its pathology to faulty versions of a particular protein. William Kaelin was trying to figure out how defects in this VHL protein cause the illness. The classic VHL tumor is composed of inappropriate, newly formed blood vessels, and surplus VEGF characterizes these growths; excessive erythropoietin production also can occur. Kaelin wondered whether VHL influences activity of the genes for these and other hypoxic-induced substances. 

In 1996, he and his colleagues grew human cells with and without operational VHL. Then they measured the abundance of several messenger RNAs, including VEGF’s, that normally disappear in response to oxygen. Even when oxygen was plentiful, VHL-defective cells contained large amounts of these mRNAs. Addition of intact VHL restored normal hypoxia-dependent quantities. 

Kaelin then showed that VHL’s capacity to quash the accumulation of particular mRNAs in rich oxygen conditions relies on its ability to physically assemble with several other proteins, including one that was later shown to mark certain proteins for destruction by attaching the chemical tag ubiquitin. Ubiquitin is the signal that sends certain proteins to the cell’s incinerator, the proteasome (Albert Lasker Basic Medical Research Award, 2000). Research soon established that HIF-1α is degraded through the ubiquitin pathway when oxygen is profuse. 

In 1999, Ratcliffe and colleagues demonstrated that the oxygen-dependent elimination of HIF-1α depends on VHL. Furthermore, under conditions that jam the proteasome, VHL and HIF-1α are present in the same protein assembly. Ratcliffe proposed that VHL interacts with HIF-1α under high-oxygen conditions and targets it for destruction. 

Kaelin subsequently found that VHL binds directly to HIF-1α—and that optimal binding requires a region in HIF-1α that was known to be needed for its oxygen-triggered destruction. He, Ratcliffe, and others showed that the same region is ubiquitinated and that defects in VHL prevent addition of the chemical tag. Together, these observations established that the VHL assembly directs ubiquitination of HIF-1α when oxygen is present. 

A crucial oxygen-based modification

Ratcliffe and Kaelin wondered what allows VHL to bind HIF-1α under high- but not low-oxygen conditions. Independently, they used various tricks to block the ubiquitin pathway, thereby allowing HIF-1α to accumulate even when oxygen abounds. Their observations suggested the existence of an enzyme that modifies HIF-1α such that it can snag VHL. 

To identify the presumptive HIF-1α modification, both teams homed in on the region of HIF-1α that grabs VHL. Elimination of a single amino acid—a proline—in this region abrogates VHL’s ability to attach ubiquitins and thus safeguards HIF-1. Further analysis revealed that this proline had picked up an oxygen atom next to one of its hydrogens. HIF-1α had thus acquired a chemical modification called a hydroxyl group. 

Together, these and other findings indicated that a prolyl hydroxylase, named for its deeds, adds a hydroxyl to a critical proline in HIF-1α, thereby rendering HIF-1α recognizable by VHL and fostering its consequent ruin. Because prolyl hydroxylases require molecular oxygen to do their jobs, the observations, published in 2001, explained why HIF-1α is not degraded under hypoxic conditions and thus, how the enzyme translates oxygen levels into HIF-1α stability (see figure).

The experiments had not, however, uncovered the specific prolyl hydroxylase that acts on HIF-1α, and the amino acid sequence around the hydroxylated HIF-1α proline did not match target sites for known prolyl hydroxylases. In collaboration with Christopher Schofield (University of Oxford), Ratcliffe proposed that the enzyme belongs to a larger molecular family. Ratcliffe and, independently, Steven McKnight (University of Texas Southwestern Medical Center) identified three related prolyl hydroxylases that govern the cellular response to oxygen in mammals.

Ratcliffe subsequently demonstrated that hydroxylation of another proline in HIF-1α also promotes its oxygen-dependent, VHL-mediated demolition. Soon afterward, others found that hydroxylation of a different amino acid incapacitates HIF-1α as well, but in a different way. 

Breathing new life into therapies

We now know that HIF-1 and its molecular relatives regulate a plethora of genes whose products influence a vast number of biological processes. Harnessing the HIF pathway for therapeutic purposes hence offers fertile potential. 

For example, prolyl hydroxylase inhibitors that preserve HIF and thus incite erythropoietin gene activity hold hope of reversing anemia. Results in animals as well as humans show promise, and several companies are pursuing this possibility. Interfering with HIF prolyl hydroxylases might also promote blood-vessel growth and other adaptations to hypoxia that would combat conditions resulting from inadequate circulation.

On the flip side, thwarting HIF might quell malignancies, in part because many tumors draw nourishment from extra blood vessels they construct. A compound that foils HIF-2α, a close relative of HIF-1α, is currently in early clinical trials for kidney cancer.  

Kaelin, Ratcliffe, and Semenza have unveiled the fundamental workings of HIF, which presides over a complex and exquisitely controlled response to oxygen fluctuations. Their discoveries have revealed secrets about a cornerstone of life on Earth.

by Evelyn Strauss

Key Publications of William G. Kaelin, Jr.

Iliopoulos, O., Levy, A.P., Jiang, C., Kaelin, W.G., Jr., and Goldberg, M.A. (1996). Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc. Natl. Acad. Sci. USA. 93, 10595-10599.  

Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.-Y., Huang, L.E., Chau, V., Pavletich, N., and Kaelin, W.G., Jr. (2000). Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423-427. 

Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S., and Kaelin, W.G., Jr. (2001). HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O₂ sensing. Science. 292, 464-468. 

Ivan, M., Haberberger, T., Gervasi, D., Michelson, K.S., Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R.C., Conaway, J.W., and Kaelin, W.G., Jr. (2002). Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc. Natl. Acad. Sci. USA. 99, 13459-13464. 

Kaelin, W.G., Jr. and Ratcliffe, P.J. (2008). Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol. Cell. 4, 393-402.

Key Publications of Peter J. Ratcliffe

Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (1993). Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc. Natl. Acad. Sci. USA. 90, 2423-2427.

Maxwell, P.H., Wiesener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R., and Ratcliffe, P.J.  (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 399, 271-275.

Jaakkola, P., Mole, D.R., Tian, Y.-M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (2001). Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O₂-regulated prolyl hydroxylation. Science. 292, 468-472.

Epstein, A.C.R., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O’Rourke, J.F., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., Tian, Y.-M., Masson, N., Hamilton, D.L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P.H., Pugh, C.W., Schofield, C.J., and Ratcliffe, P.J. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 107, 43-54.

Cockman, M.E., Lancaster, D.E., Stolze, I.P., Hewitson, K.S., McDonough, M.A., Coleman, M.L., Coles, C.H., Yu, X., Hay, R.T., Ley, S.C., Pugh, C.W., Oldham, N.J., Masson, N., Schofield, C.J., and Ratcliffe, P.J. (2006). Post-translational hydroxylation of ankyrin repeats in IkB proteins by the HIF asparaginyl hydroxylases, FIH. Proc. Natl. Acad. Sci. USA 103, 14767-14722.

Key Publications of Gregg L. Semenza

Semenza, G.L., Nejfelt, M.K., Chi, S.M., and Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer located 3' to the human erythropoietin gene. Proc. Natl. Acad. Sci. USA. 88, 5680-5684.

Semenza, G.L. and Wang, G.L. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol. 12, 5447-5454.

Wang, G.L. and Semenza, G.L. (1995). Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270, 1230-1237.

Wang, G.L., Jiang, B.-H., Rue, E.A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O₂ tension. Proc. Natl. Acad. Sci. USA. 92, 5510-5514.

Forsythe, J.A., Jiang, B.-H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., and Semenza, G.L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604-4613.

Craig Thompson Commentary in Cell

Oxygen is carried by red blood cells.  If red blood cells are deficient, oxygen transport fails, and cells die.  So animals evolved a system to detect oxygen deficiency and to increase red blood cell production. The sensor is located in the kidney.  When deprived of oxygen, the kidney secretes a protein called EPO that travels to the bone marrow where it stimulates production of new red blood cells.   EPO was purified in 1977 by the late Eugene Goldwasser.  It took him 15 years.  With the protein sequence in hand, the EPO gene was cloned in 6 months.  The fundamental question was exposed.  How do kidney cells activate the EPO gene in response to oxygen deficiency?  This is not a simple problem.  Oxygen is a gas.  How do cells measure a gas, and how do they use this information to activate a gene?

Mother Nature is a strip-tease artist.  She reveals herself in stages.  To expose her beauty scientists must remove layer after layer. Greg Semenza removed Mother Nature's coat.   In the early 90's he guessed that the EPO gene works like other genes.  There must be a protein that activates it.   The protein must bind to a piece of DNA near the gene called an enhancer.  Semenza identified the enhancer.  He used the enhancer as bait to purify the transcription factor and he named it hypoxia inducible factor or HIF.  He found that the amount of HIF increases when cells are deprived of oxygen. HIF enters the nucleus where it activates the EPO gene.

Mother Nature’s coat was gone.  Next came her dress. The riddle was: "how does oxygen deprivation increase HIF?  This is where our other two winners came in.    Working almost in tandem Ratcliffe and Kaelin identified the protein that regulates HIF. The protein has a hybrid German-Swedish name: Von Hippel Lindau. We will call it VHL.  VHL is a HIF executioner. It binds to HIF and destroys it.  When VHL is active each HIF molecule lives only a few minutes - far too short to activate the EPO gene. Somehow a deficiency of oxygen must silence VHL so that HIF can survive long enough to enter the nucleus.

The discovery of VHL removed Mother Nature's dress.  The end was near. But first Drs. Ratcliffe and Kaelin had to figure out how oxygen inactivates VHL.  The key is the way in which VHL recognizes HIF.  VHL binds to amino acids on HIF that have oxygen atoms attached to them. If these amino acids lack oxygen, VHL cannot destroy HIF and HIF will live to activate EPO.  What attaches oxygen to HIF?  There must be an enzyme.  The race to find the enzyme was on.  Three teams crossed the finish line at once. They were led by Kaelin, Ratliffe and a late entry in the game – Steve McKnight of UT Southwestern in Dallas. The enzymes are called dioxygenases.  They latch onto oxygen and transfer it to HIF.  Oxygen-coated HIF is recognized by VHL and destroyed.  When oxygen levels are low the dioxygenases have nothing to transfer. HIF lacks oxygen, and it escapes VHL.  HIF enters the nucleus and activates the EPO gene.  At last the problem was solved.  Nature’s beauty was revealed.

All great strip-tease artists save a pleasant surprise for the end, and Mother Nature is no exception.  Her surprise is a cure for cancer.  The search for an oxygen sensor began as a quest for an activator of the EPO gene.  Nature’s surprise was that HIF activates much more than EPO. It activates 200 other genes.  Moreover, HIF is not confined to the kidney.  HIF is found in all cells.  What other genes does HIF activate?  Some of them increase oxygen delivery by stimulating growth of blood vessels. HIF is essential for cancers that need new blood vessels to support growth. HIF also activates genes called cyclins that cause cancer cells to grow. High levels of HIF can be tragic. Rare people inherit mutations in VHL.  Without VHL, their cells cannot destroy HIF.  Unrestrained HIF induces tumors in the eye, in the nervous system and in the kidney. Even when people don’t inherit mutations in VHL their kidney cells can acquire mutations and the elevated HIF drives tumor formation.  Recently, a biotech company has invented an inhibitor of HIF and it shows early promise in the treatment of patients with kidney cancer.

My strip-tease analogy is not perfect.  Strip tease artists remove their own clothes.  Mother Nature needs help.  Passionate scientists must fumble with the buttons, zippers and hooks.  Doctors Semenza, Ratliffe and Kaelin didn’t fumble.  Guided by brilliant intuition and powered by delicate technology they deftly removed each layer.   I can only imagine their delight as each discovery moved them closer to the ultimate answer.  These three doctors exposed the beauty of Mother Nature, and they clearly deserve the Albert and Mary Lasker Basic Science Award.

As a premed, I floundered in a research laboratory working on an independent study project that was uninteresting, unimportant, and, as I would later correctly show, undoable.  My mentor rewarded me with a “C+,” noting on my transcript that “Mr. Kaelin appears to be a bright young man whose future lies outside of the laboratory.” This painful experience convinced me I was going to be a clinician rather than a scientist, and I approached my clinical training accordingly.  I even served as Chief Resident at Johns Hopkins, honing my knowledge of obscure diseases such as von Hippel-Lindau Disease.

Years later, I discovered, thanks to outstanding mentorship from David Livingston, that I actually could do science.   And my clinical practice convinced me that we desperately needed better cancer treatments based on a deeper understanding of cancer pathogenesis.

I like mathematics, medicine, and science because I like solving puzzles, and I like answers that are objectively verifiable.  Science gathers knowledge, and engineering applies that knowledge to useful purposes.   For most of my career, I benefited from the wise decision in our country to have the public sector support basic scientific research, where the timelines and deliverables are too unpredictable for investors, and to let the private sector decide when a line of investigation is ripe for application and commercialization.   Although this bargain served American biomedical research well, one hears repeated calls to treat science as though it were engineering, tying funding to anticipated outcomes and impact.  This is troubling because many practices that are useful for managing engineering projects are antithetical to good science.  For example, building teams and harmonizing goals is often essential for large engineering projects.  Early stage science, however, is often driven by creative individuals who follow their curiosity and are willing to go where their science takes them.    Forcing scientists into teams and holding them to predetermined deliverables can create herd mentality and stifle the heretical thinking that is often needed for transformative discoveries.

JFK knew it would take a decade to put a man on the moon because it was fundamentally an engineering challenge rather than a scientific challenge. It does scientists and, more importantly, patients and their families, a disservice when fundraisers and policy makers overpromise and oversimplify with respect to our greatest biomedical challenges, including cancer.

I am very proud to receive this award on behalf of the talented young scientists who have worked in my laboratory over the years, to share it with such esteemed colleagues, and to dedicate it to my beloved wife Carolyn, who died last summer. 

Key Publications of William G. Kaelin, Jr.

Iliopoulos, O., Levy, A.P., Jiang, C., Kaelin, W.G., Jr., and Goldberg, M.A. (1996). Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc. Natl. Acad. Sci. USA. 93, 10595-10599.  

Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.-Y., Huang, L.E., Chau, V., Pavletich, N., and Kaelin, W.G., Jr. (2000). Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423-427. 

Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S., and Kaelin, W.G., Jr. (2001). HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O₂ sensing. Science. 292, 464-468. 

Ivan, M., Haberberger, T., Gervasi, D., Michelson, K.S., Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R.C., Conaway, J.W., and Kaelin, W.G., Jr. (2002). Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc. Natl. Acad. Sci. USA. 99, 13459-13464. 

Kaelin, W.G., Jr. and Ratcliffe, P.J. (2008). Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol. Cell. 4, 393-402.

I did train in medicine, as a nephrologist, and came to research rather late, fascinated by the extraordinary sensitivity with which the kidneys regulate the hormone erythropoietin in accordance with blood oxygen content. I felt that the process was interesting and might be tractable. At the time, the erythropoietin gene had recently been identified, so there was a new opportunity for study.  But some felt, with the emerging success of recombinant erythropoietin treatment, that understanding how the hormone was regulated was niche area, unlikely to be of general importance.

Key Publications of Peter J. Ratcliffe

Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (1993). Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc. Natl. Acad. Sci. USA. 90, 2423-2427.

Maxwell, P.H., Wiesener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R., and Ratcliffe, P.J.  (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 399, 271-275.

Jaakkola, P., Mole, D.R., Tian, Y.-M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (2001). Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O₂-regulated prolyl hydroxylation. Science. 292, 468-472.

Epstein, A.C.R., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O’Rourke, J.F., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., Tian, Y.-M., Masson, N., Hamilton, D.L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P.H., Pugh, C.W., Schofield, C.J., and Ratcliffe, P.J. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 107, 43-54.

Cockman, M.E., Lancaster, D.E., Stolze, I.P., Hewitson, K.S., McDonough, M.A., Coleman, M.L., Coles, C.H., Yu, X., Hay, R.T., Ley, S.C., Pugh, C.W., Oldham, N.J., Masson, N., Schofield, C.J., and Ratcliffe, P.J. (2006). Post-translational hydroxylation of ankyrin repeats in IkB proteins by the HIF asparaginyl hydroxylases, FIH. Proc. Natl. Acad. Sci. USA 103, 14767-14722.

Eight months ago, David Bowie died of liver cancer. Cancer cells invade surrounding tissue, make their way into blood vessels, and spread throughout the body. What are they searching for? My guess is oxygen.

Over the last 25 years, we’ve worked to understand how a family of proteins, which we called hypoxia-inducible factors, controls the ability of cells, tissues, and organ systems to respond to changes in oxygen availability.  Our work began with an attempt to figure out how expression of the erythropoietin gene was turned on when the body was deprived of oxygen. Now we know that more than 2,000 other genes are regulated in a similar manner.

Looking forward, I’d like to think that within the next decade drugs that stimulate hypoxia-inducible factors will be used to treat anemia and cardiovascular disease, while drugs that inhibit these factors will prolong the lives of cancer patients.

So this is my religion:  I am filled with Wonder at the outcome of 4 billion years of evolution here on our speck in the universe and Hope regarding our opportunity to improve the lives of those around us through basic science discoveries and their translation to clinical practice.

Well, I began with Mary Lasker, so I’ll end with Albert. As you know, Mr. Lasker was a successful advertising executive. He was in charge of accounts for products like Sunkist oranges and Pepsodent toothpaste before he and Mary began their crusade to promote biomedical research. Coincidentally, my grandfather also worked for an advertising agency here in Manhattan. His account was for a product called Wonder Bread.

Thank you.

Key Publications of Gregg L. Semenza

Semenza, G.L., Nejfelt, M.K., Chi, S.M., and Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer located 3' to the human erythropoietin gene. Proc. Natl. Acad. Sci. USA. 88, 5680-5684.

Semenza, G.L. and Wang, G.L. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol. 12, 5447-5454.

Wang, G.L. and Semenza, G.L. (1995). Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270, 1230-1237.

Wang, G.L., Jiang, B.-H., Rue, E.A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O₂ tension. Proc. Natl. Acad. Sci. USA. 92, 5510-5514.

Forsythe, J.A., Jiang, B.-H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., and Semenza, G.L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604-4613.