Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of development and 
function of digestive tissues. The HNF4A gene uses two separate promoters P1 and 
P2, with P1 products predominant in adult liver, whereas P2 products prevalent 
in fetal liver, pancreas, and liver/colon cancer. To date, the mechanisms for 
the regulation of HNF4A and the dynamic switch of P1-HNF4α and P2-HNF4α during 
ontogenesis and carcinogenesis are still obscure. Our study validated the 
previously reported self-stimulation of P1-HNF4α but invalidated the reported 
synergism between HNF4α and HNF1α. HNF4A-AS1, a long noncoding RNA, is localized 
between the P2 and P1 promoters of HNF4A. We identified critical roles of 
P1-HNF4α in regulating the expression of HNF4A-AS1 and its mouse ortholog 
Hnf4a-os. Paired box 6 (PAX6), a master regulator of pancreas development 
overexpressed in colon cancer, cooperated with HNF1α to induce P2-HNF4α but 
antagonized HNF4α in HNF4A-AS1 expression. Thus, PAX6 may be important in 
determining ontogenic and carcinogenic changes of P2-HNF4α and HNF4A-AS1 in the 
pancreas and intestine. We also interrogated transactivation activities on 
multiple gene targets by multiple known and novel HNF4α mutants identified in 
patients with maturity onset diabetes of the young 1 (MODY1) and liver cancer. 
Particularly, HNF4α-D78A and HNF4α-G79S, two mutants found in liver cancer with 
mutations in DNA-binding domain, displayed highly gene-specific transactivation 
activities. Interestingly, HNF4α-Q277X, a MODY1 truncation mutant, antagonized 
the transactivation activities of HNF1α and farnesoid X receptor, key regulators 
of insulin secretion. Taken together, our study provides novel mechanistic 
insights regarding the transcriptional regulation and transactivation activity 
of HNF4α in digestive tissues.